Survivin-derived peptides and uses thereof

ABSTRACT

MHC Class I-restricted peptides derived from the tumor associated antigen, survivin, which peptides are capable of binding to Class I HLA molecules at a high affinity, capable of eliciting INF-γ-producing cells in a PBL population of a cancer patient and capable of in situ detection of cytotoxic T cells in a tumor tissue, therapeutic and diagnostic composition comprising the peptide and uses thereof.

This application is a divisional patent application of U.S. patentapplication Ser. No. 10/543,755 filed Jun. 1, 2006, now U.S. Pat. No.8,318,174, which is a 371 national stage filing of PCT/DK2004/000062,filed Jan. 30, 2004, which is a continuation of U.S. patent applicationSer. No. 10/354,090, filed Jan. 30, 2004, now abandoned, and which isalso a continuation of U.S. patent application Ser. No. 10/715,417,filed Nov. 19, 2003, now U.S. Pat. No. 7,892,559, which is acontinuation-in-part of U.S. application Ser. No. 10/354,090, filed Jan.30, 2003, now abandoned, which claims priority to U.S. ProvisionalPatent Application No. 60/352,284, filed Jan. 30, 2002, the disclosuresof each of which are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to novel survivin-derived peptides andtheir use for diagnostic and therapeutic purposes, specifically incancer. In particular, the novel peptides are MHC Class I-restrictedT-cell epitopes that are capable of eliciting cytotoxic T-cell responsesin cancer patients including in situ and ex vivo responses.Specifically, such novel peptides are derived from the apoptosisinhibitor protein survivin, a recognized tumor associated antigen (TAA).

TECHNICAL BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T-cell response. This response requires that T cellsrecognize and interact with complexes of cell surface molecules referredto as human leukocyte antigens (HLA) constituting the human majorhistocompatibility complex (MHC), and peptides. The peptides are derivedfrom larger molecules, which are processed by the cells, which alsopresent the HLA/MHC molecule. The interaction of T cells and complexesof HLA/peptide is restricted, requiring a T cell that is specific for aparticular combination of an HLA molecule and a peptide. If a specific Tcell is not present, there is no T-cell response even if its partnercomplex is present. Similarly, there is no response if the specificcomplex is absent, but the T cell is present.

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. E.g. in WO92/20356, a family of genes isdisclosed which are processed into peptides which, in turn, areexpressed on cells surfaces, and can lead to lysis of the tumour cellsby specific CTLs. These genes are referred to as the MAGE family and aresaid to code for “tumour rejection antigen precursors” or “TRAP”molecules, and the peptides derived therefrom are referred to as “tumourrejection antigens” or “TRAs”.

In WO 94/05304, nonapeptides are disclosed which bind to the HLA-A1molecule. The reference discloses that given the known specificity ofparticular peptides for particular HLA molecules, one should expect aparticular peptide to bind one HLA molecule, but not others. This issignificant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype.

Several peptides presented by MHC molecules have been characterized andit has been found that some of these may carry posttranslationalmodifications possibly having an impact on the functionality of theHLA-peptide complex. Thus, a number of studies have associatedalterations in the pattern of phosphorylation with malignanttransformation. Furthermore, it has been shown that phosphorylationcould have a neutral, negative or even a positive effect on peptidebinding to class I MHC and that phosphopeptide-specific CTL, whichdiscriminated between the phosphorylated and the non-phosphorylatedversions of the peptide, could be generated, showing that such CTL mostlikely are part of the class I MHC-restricted CTL repertoire. Recently,it has been shown that phosphorylated peptides indeed are processednaturally and presented by MHC class I molecules in vivo. Additionally,the presence of phosphorylated peptides in extracts from isolated classI molecules from several different EBV-transformed B-cells has beenestablished.

Thus, it is well established that peptide epitopes derived from tumorassociated antigens (TAAs) can be recognized as antigens by cytotoxic Tlymphocytes (CTLs) in the context of MHC molecules (1). However,although it is generally accepted that most if not all, tumours areantigenic, only a few are indeed immunogenic in the sense that tumourprogression is readily controlled by the immune system.

To overcome this limitation, several immunotherapeutic trials have beeninitiated, e.g. vaccinations with TAA-derived peptides. For melanoma,the tumour for which the largest number of CTL-defined TAAs has beencharacterized, powerful CTL responses against antigens have been inducedby vaccination and some patients experienced a complete remission oftheir disease (2,3). However, most of the peptide epitopes used in thesevaccination trials are melanocyte specific, and these peptides cannot beapplied for tumours of non-melanocyte origin. Furthermore, expression ofthese TAAs is heterogeneous among tumours from different patients andcan even vary among metastases obtained from one patient. However,during the last couple of years a number of tumour specific peptideantigens, which are expressed in a number of different cancers, havebeen identified, i.e. HER-2 (4), Muc-1 (5) and telomerase (6).

It has also been shown that by proper manipulation tumor antigenspresent in tumors can be exposed to the immune system. Studies haveshown that the CD8+ CTL arm of the immune response, alone or incombination with CD4+ T_(h) cells, constitutes the primary anti-tumoreffector arm of the adaptive immune response. Up till now the focus hasmainly been on the CTL arm of the immune response. However, it isbecoming more and more clear that the CD4 T cell response plays anessential role in tumor rejection, especially in the induction phase orin the extension of a CTL response in vivo. Consequently, theincorporation of class II-restricted tumor antigens into effective tumorvaccination protocols might increase the effectiveness of the vaccines.

Apoptosis is a genetic program of cellular suicide, and Inhibition ofapoptosis has been suggested to be an important mechanism involved incancer formation by extending the life span of cells favouring theaccumulation of transforming mutations (7). Survivin is a recentlyidentified member of the family of inhibitors of apoptosis proteins(IAPs). In a global gene expression analysis of about 4 milliontranscripts, survivin was identified as one of the top genes invariablyup-regulated in many types of cancer but not in normal tissue (8). Solidmalignancies overexpressing survivin include lung, colon, breast,pancreas, and prostate cancer as well as hematopoietic malignancies (9).Additionally, series of melanoma and non-melanoma skin cancers have beenreported to be invariably survivin positive (10,11). The overexpressionof survivin in most human cancers suggests a general role of apoptosisinhibition in tumor progression, a notion substantiated by theobservation that in the case of colorectal and bladder cancer, as wellas neuroblastoma, expression of survivin was associated with anunfavourable prognosis. In contrast, survivin is undetectable in normaladult tissues. These characteristics qualify survivin as a suitable TAAfor both diagnostic and therapeutic purposes.

Thus, during the last decade a large number of TAAs have been identifiedwhich are recognized by CTLs in a major histocompatibility complex(MHC)-restricted fashion. As survivin is overexpressed in most humancancers and inhibition of its function results in increased apoptosis,this protein may serve as a target for therapeutic CTL responses. Thesurvivin protein and the potential diagnostic and therapeutic use hereofare disclosed in (8) and U.S. Pat. No. 6,245,523, which are incorporatedherein by reference. Survivin is a 16.5 kDa cytoplasmic proteincontaining a single BIR and a highly charged carboxy-terminal coiledregion instead of a RING finger, which inhibits apoptosis induced bygrowth factor (IL-3) withdrawal when transferred in B cell precursors.The gene coding for survivin is nearly identical to the sequence ofEffector Cell Protease Receptor-1 (EPR-1), but oriented in the oppositedirection, thus suggesting the existence of two separate genesduplicated in a head-to-head configuration. Accordingly, survivin can bedescribed as an antisense EPR-1 product. Functionally, inhibition ofsurvivin expression by up-regulating its natural antisense EPR-1transcript results in massive apoptosis and decreased cell growth.

U.S. Pat. No. 6,245,523 discloses the isolation of purified survivin andit provides nucleic acid molecules that encode the survivin protein, andantibodies and other molecules that bind to survivin. U.S. Pat. No.6,245,523 also discloses anti-apoptotically active fragments of thesurvivin protein and variants hereof wherein an amino acid residue hasbeen inserted N- or C-terminal to, or within, the disclosed survivinsequence. It is specifically disclosed that such peptides should containkey functional residues required for apoptosis, i.e. Trp at position 67,Pro at position 73 and Cys at position 84.

The present invention is based on the discovery that MHC Class Irestricted peptides can be derived from the survivin protein, which arecapable of binding to MHC Class I HLA molecules and thereby elicitingboth ex vivo and in situ CTL immune responses in patients suffering froma wide range of cancer diseases. These findings strongly suggest thatsurvivin acts as a TRAP molecule, which is processed by cells intopeptides having TRA functionality. Evidently, these findings open theway for novel therapeutic and diagnostic approaches which, due to thefact that survivin appears to be expressed universally by tumour cells,are generally applicable in the control of cancer diseases.

SUMMARY OF THE INVENTION

Accordingly, the invention pertains in a first aspect to a MHC ClassI-restricted epitope peptide derived from survivin, said epitope havingat least one of the following characteristics:

(i) capable of binding to the Class I HLA molecule to which it isrestricted at an affinity as measured by the amount of the peptide thatis capable of half maximal recovery of the Class I HLA molecule (C₅₀value) which is at the most 50 μM as determined by the assembly bindingassay as described herein,

(ii) capable of eliciting INF-γ-producing cells in a PBL population of acancer patient at a frequency of at least 1 per 10⁴ PBLs as determinedby an ELISPOT assay, and/or

(iii) capable of in situ detection in a tumour tissue of CTLs that arereactive with the epitope peptide.

Preferably, the peptide of the invention has at least two, mostpreferably all of these three features.

In further aspects the invention provides a pharmaceutical compositionand a composition for ex vivo or in situ diagnosis of the presence in acancer patient of survivin reactive T-cells among PBLs or in tumourtissue, which composition comprises a peptide as defined above.

In yet further aspects the invention relates to a diagnostic kit for exvivo or in situ diagnosis of the presence in a cancer patient ofsurvivin reactive T-cells among PBLs or in tumor tissue, which kitcomprises a peptide according of the invention, and a complex of such apeptide and a Class I HLA molecule or a fragment of such molecule.

In another aspect there is also provided a method of detecting in acancer patient the presence of survivin reactive T-cells, the methodcomprising contacting a tumour tissue or a blood sample with a complexas defined above and detecting binding of the complex to the tissue orthe blood cells.

In still further aspects the invention pertains to a molecule that iscapable of binding specifically to a peptide of the invention such as anantibody or a fragment hereof, and to a molecule that is capable ofblocking the binding of such a molecule.

An important aspect of the invention relates to the use of the peptidesof the invention for the preparation of a medicament for the treatmentof cancer. A further aspect relates to the use of the composition or themolecule as mentioned above for the preparation of a medicament for thetreatment of cancer.

Still further aspects relate independently to a method for treatingcancer in a mammal, such as a human, comprising the administration to apatient suffering from the disease an effective amount of the peptide,composition or a molecule of the invention.

DETAILED DISCLOSURE OF THE INVENTION

The novel MHC Class I-restricted peptide of the invention ischaracterised by having at least one of several features, one of whichis the ability to bind to the Class I HLA molecule to which it isrestricted at an affinity, which, when it is measured by the amount ofthe peptide that is capable of half maximal recovery of the Class I HLAmolecule (C₅₀ value) in an assembly assay as described herein, is at themost 50 μM. This assembly assay is carried out as described previously(12,13), and it is based on stabilisation of the HLA molecule afterloading of peptide to the peptide transporter deficient cell line T2.Subsequently, correctly folded stable HLA heavy chains areimmunoprecipitated using conformation dependent antibodies and thepeptide binding is quantitated.

This assay provides a simple means of screening candidate peptides fortheir ability to bind to a given HLA allele molecule at the aboveaffinity. In preferred embodiments, the peptide of the invention In onehaving a C₅₀ value, which is at the most 30 μM, such as a C₅₀ value,which is at the most 20 μM including C₅₀ values of at the most 10 μM, atthe most 5 μM and at the most 2 μM.

As mentioned above, the HLA system represents the human majorhistocompatibillty (MHC) system. Generally, MHC systems control a rangeof characteristics: transplantation antigens, thymus dependent immuneresponses, certain complement factors and predisposition for certaindiseases. More specifically, the MHC codes for three different types ofmolecules, i.e. Class I, II and III molecules, which determine the moregeneral characteristics of the MHC. Of these molecules, the Class Imolecules are so-called HLA-A, HLA-B and HLA-C molecules that arepresented on the surface of most nucleated cells and thrombocytes.

The peptides of the present invention are characterised by their abilityto bind to (being restricted to) a particular MHC Class I HLA molecule.Thus, in one embodiment the peptide is one which is restricted to a MHCClass I HLA-A molecule including HLA-A1, HLA-A2, HLA-A3, HLA-A9,HLA-A10, HLA-A11, HLA-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10),HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31(w19),HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43,HLA-Aw66(10), HLA-Aw68(28), HLA-A69(28). More simple designations arealso used throughout the literature, where only the primary numericdesignation is used, e.g. HLA-A19 or HLA-A24 instead of HLA-Aw19 andHLA-A24(9), respectively. In specific embodiments, the peptide of theinvention is restricted to a MHC Class I HLA species selected from thegroup consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.

The peptides of the invention are derived from the known sequence ofsurvivin, e.g. the sequence disclosed in U.S. Pat. No. 6,245,523. Theselection of peptides potentially having the ability to bind to aparticular HLA molecule can be made by the alignment of known sequencesthat bind to a given particular HLA molecule to thereby reveal thepredominance of a few related amino acids at particular positions in thepeptides. Such predominant amino acid residues are also referred toherein as “anchor residues” or “anchor residue motifs”. By followingsuch a relatively simple procedure based on known sequence data that canbe found in accessible databases, peptides can be derived from thesurvivin protein molecule which are likely to bind to the particular HLAmolecule. Representative examples of such analyses for a range of HLAmolecules are given in the below table:

HLA C- allele Position 1 Position 2 Position 3 Position 5 Position 6Position 7 terminal HLA-A1 T, S D, E L Y HLA-A2 L, M V L, V HLA-A3 L, V,M F, Y K, Y, F HLA-A11 V, I, F, Y M, L, F, Y, I K, R HLA-A23 I, Y W, IHLA-A24 Y I, V F I, L, F HLA-A25 M, A, T I W HLA-A26 E, D V, T, I, L, FI, L, V Y, F HLA-A28 E, D V, A, L A, R HLA-A29 E Y, L HLA-A30 Y, L, F, VY HLA-A31 L, M, F, Y R HLA-A32 I, L W HLA-A33 Y, I, L, V R HLA-A34 V, LR HLA-A66 E, D T, V R, K HLA-A68 E, D T, V R, K HLA-A69 V, T, A V, LHLA-A74 T V, L HLA-B5 A, P F, Y I, L HLA-B7 P L, F HLA-B8 K K, R LHLA-B14 R, K L, V HLA-B15 Q, L, K, P, H, F, Y, W (B62) V, I, M, S, THLA-B17 L, V HLA-B27 R Y, K, F, L HLA-B35 P I, L, M, Y HLA-B37 D, E I,L, M HLA-B38 H D, E F, L HLA-B39 R, H L, F HLA-B40 E F, I, V L, V, A, W,M, (B60,61) T, R HLA-B42 L, P Y, L HLA-B44 E F, Y, W HLA-B46 M, I, L, VY, F HLA-B48 Q, K L HLA-B51 A, P, G F, Y, I, V HLA-B52 Q F, Y I, VHLA-B53 P W, F, L HLA-B54 P HLA-B55 P A, V HLA-B56 P A, V HLA-B57 A, T,S F, W, Y HLA-B58 A, T, S F, W, Y HLA-B67 P L HLA-B73 R P HLA-Cw1 A, L LHLA-Cw2 A, L F, Y HLA-Cw3 A, L L, M HLA-Cw4 Y, P, F L, M, F, Y HLA-Cw6 YL, Y, F, Y HLA-Cw8 Y L, I, HLA-Cw16 A, L L, V

Thus, as an example, nonapeptides potentially having the ability to bindto HLA-A1 would have one of the following sequences:Xaa-T-D-Xaa-Xaa-Xaa-L-Xaa-Y, Xaa-T-E-Xaa-Xaa-Xaa-L-Xaa-Y;Xaa-S-D-Xaa-Xaa-Xaa-L-Xaa-Y or Xaa-S-E-Xaa-Xaa-Xaa-L-Xaa-Y (Xaaindicating any amino acid residue). In a similar manner, sequencespotentially having the ability to bind to any other HLA molecule can bedesigned.

It will be appreciated that the person of ordinary skill in the art willbe able to identify further “anchor residue motifs” for a given HLAmolecule.

Thus, in useful embodiments, the peptides of the invention includepeptides, the sequences of which comprise, for each of the specific HLAalleles listed in the table, any of the amino acid residues as indicatedin the table.

Thus, a simple approach to identifying peptides of the inventionincludes the following steps: selecting a particular HLA molecule, e.g.one occurring at a high rate in a given population, carrying out analignment analysis as described above to identify “anchor residuemotifs” in the survivin protein, isolating or constructing peptides of asuitable size that comprise one or more of the identified anchorresidues and testing the resulting peptides for (i) capability to bindto the particular HLA molecule using the assembly assay as describedherein, (ii) the capability of the peptides to elicit INF-γ-producingcells in a PBL population of a cancer patient at a frequency of at least1 per 10⁴ PBLs as determined by an ELISPOT assay as described herein,and/or (iii) the capability of the peptides to detect in situ in atumour tissue CTLs that are reactive with the epitope peptides beingtested.

In specific embodiments, the peptide of the invention is an HLA-A2restricted survivin-derived peptide having a sequence selected from thefollowing: FLKLDRERA (survivin₁₀₁₋₁₀₉) (SEQ ID NO:1), TLPPAWQPFL(survivin₅₋₁₄) (SEQ ID NO:2), ELTLGEFLKL (survivin₉₅₋₁₀₄) (SEQ ID NO:3),LLLGEFLKL (SEQ ID NO:4) and LMLGEFLKL (SEQ ID NO:5). (The designationsin brackets indicate the positions of the residues in the survivinprotein as disclosed in U.S. Pat. No. 6,245,523). LLLGEFLKL (SEQ IDNO:4) is a sequence derived from survivin₉₆₋₁₀₄ by substituting “T” inposition 2 of the peptide with an “L” and LMLGEFLKL (SEQ ID NO:5) isderived from survivin₉₆₋₁₀₄ by substituting “T” in position 2 with “M”.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class I HLA-B molecule includingany of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14,HLA-B15, HLA-B16, HLA-817, HLA-B18, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35,HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44,HLA-B45, HLA-Bw46 and HLA-Bw47. In specific embodiments, the MHC Class IHLA-B species to which the peptide of the invention is capable ofbinding is selected from HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15,HLA-B27 and HLA-B51.

In specific embodiments, the peptide of the invention is anHLA-B35-restricted survivin-derived peptide having a sequence selectedfrom the following: CPTENEPDL (survivin₄₆₋₅₄) (SEQ ID NO:6), EPDLAQCFF(survivin₅₁₋₅₉) (SEQ ID NO:7), CPTENEPDY (SEQ ID NO:8) and EPDLAQCFY(SEQ ID NO:9). (The designations in brackets indicate the positions ofthe residues in the survivin protein as disclosed in U.S. Pat. No.6,245,523). CPTENEPDY (SEQ ID NO:8) is a sequence derived fromsurvivin₄₆₋₅₄ by substituting “L” in the C-terminal of the peptide witha “Y” and EPDLAQCFY (SEQ ID NO:9) is derived from survivin₅₁₋₅₉ bysubstituting an “F” residue in the C-terminal 2 with a “Y”.

In further specific embodiments, the peptide of the invention is aHLA-A1 restricted peptide having a sequence selected from the following:Survivin₃₈₋₄₆ (Sur38Y9) (a C changed to a Y at P9, MAEAGFIHY) (SEQ IDNO:38), Survivin₄₇₋₅₆ (Sur47Y10) (a Q changed to a Y at P10, PTENEPDLAY(SEQ ID NO:39)), Survivin₉₂₋₁₀₁ (Sur92-101) (QFEELTLGEF) (SEQ ID NO:27),and Survivin₉₃₋₁₀₁ (Sur93T2 (a E changed to a T at P2, FTELTLGEF (SEQ IDNO:36)). The peptide of the invention may also be a HLA-A3 restrictedpeptide such as Survivin₁₈₋₂₇ (Sur18K10) (a F changed to a K at P10,RISTFKNWPK (SEQ ID NO:58) and/or a HLA-A11 restricted peptide such asSurvivin₅₃₋₆₂ (Sur53-62) (DLAQCFFCFK) (SEQ ID NO:45) and/or a HLA-A2restricted peptide such as Survivin₁₈₋₂₈ (Sur18-28) (RISTFKNWPFL) (SEQID NO:66).

In further useful embodiments, the peptide of the invention is apeptide, which is restricted to a MHC Class I HLA-C molecule includingany of the following: HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5,HLA-Cw6, HLA-Cw7 and HLA-Cw16.

Preferably, the peptide of the invention comprises less than 50 aminoacid residues, and more preferably it comprises at the most 20 aminoacid residues, such as at the most 10 amino acid residues. In specificembodiments, the peptide is a heptapeptide, an octopeptide, anonapeptide, a decapeptide or an undecapeptide.

The peptide of the invention is, as mentioned above, derived from asurvivin protein or a fragment hereof. The survivin protein from whichthe peptide can be derived is survivin protein from any animal speciesin which the protein is expressed. In preferred embodiments, thesurvivin starting protein is from a mammal species including a rodentspecies, rabbit and a primate species such as humans. Based on thesequence of the selected survivin protein, the peptide of the inventionis derived by any appropriate chemical or enzymatic treatment of thesurvivin starting material that results in a peptide of a suitable sizeas indicated above, or it can be synthesised by any conventional peptidesynthesis procedures with which the person of ordinary skills in the artis familiar.

The peptide of the invention may have a sequence which is a nativesequence of the survivin protein from which is derived. However,peptides having a higher affinity to any given HLA molecule may bederived from such a native sequence by modifying the sequence bysubstituting, deleting or adding at least one amino acid residue, e.g.on the basis of the procedure described above whereby anchor residuemotifs in respect of the given HLA molecule are identified.

Accordingly, to increase the immuogenicity of survivin-derived peptides,amino acid substitutions can be introduced at anchor positions, but notat TCR contact residues, to increase peptide binding to the HLA class Imolecule. This has resulted in more immunogenic epitopes, e.g., this hasenhanced the capacity to induce cancer-reactive CTL and it has beendemonstrated to be more suitable for the induction of clinicallymeaningful CTL responses. Importantly, however, the target cancer cellsdo only express and present the native survivin-derived peptide on thecell-surface. In that respect, it is of crucial importance thattherapy-induced CTL specific for the modified survivin-derived peptidescross-react with the native analogues.

The present invention also encompasses variants and functionalequivalents of the survivin-derived peptides as disclosed herein.“Functional equivalents” as used in the present context is establishedby means of reference to the corresponding functionality of apredetermined fragment of the sequence in question. Functionalequivalence can be established by e.g. similar binding affinities to HLAclass I molecules, or similar potency demonstrated by the ELISPOT assay.

Functional equivalents or variants of a survivin-derived peptide asdescribed herein will be understood to exhibit amino acid sequencesgradually differing from the preferred, predetermined sequences, as thenumber and scope of insertions, deletions and substitutions includingconservative substitutions, increases. This difference is measured as areduction in homology between a preferred, predetermined sequence andthe survivin-derived variant or survivin-derived functional equivalent.

The homology between amino acid sequences may be calculated usingalgorithms well known in the art. Fragments sharing homology withfragments comprising or consisting of consecutive survivin-derived aminoacid residues are to be considered as falling within the scope of thepresent invention when they are preferably at least about 90%homologous, such as at least 94% homologous, including 95%, 96%, 97%,98% or 99% homologous with a predetermined survivin-derived peptide.

Furthermore, it may be advantageous to carry out post-translationalmodifications of the peptides of the invention. It has been shown thatexposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells toanticancer agents including ADRIAMYCIN® (doxorubicin), TAXOL®(paclitaxel), or UVB resulted in a 4-5-fold increased survivinexpression. Changes in survivin levels after anticancer treatment didnot involve modulation of survivin mRNA expression and were independentof de novo gene transcription. Conversely, inhibition of survivinphosphorylation on Thr³⁴ by the cyclin-dependent kinase inhibitorflavopiridol resulted in loss of survivin expression, andnonphosphorylatable survivin Thr³⁴→Ala exhibited accelerated clearanceas compared with wild-type survivin. Sequential ablation of survivinphosphorylation on Thr³⁴ enhanced tumor cell apoptosis induced byanticancer agents independently of p53 and suppressed tumor growthwithout toxicity in a breast cancer xenograft model in vivo. These datasuggest that Thr³⁴ phosphorylation critically regulates survivin levelsin tumor cells and that sequential ablation of p34^(cdc2) kinaseactivity may remove the survivin viability checkpoint and enhanceapoptosis in tumor cells.

Accordingly, it is contemplated that the survivin-derived peptides ofthe invention encompass phosphorylated peptides. Native survivinphosphopeptide antigens may be identified by scanning for the presenceof MHC peptide binding motifs around the phosphorylation site Thr34.Thus, possible survivin-derived phosphopeptide sequences Include T P E RM A E A G F, a putative HLA-B35- and/or HLA-B7- and/or aHLA-B51-restricted peptide antigen. Additional native phosphopeptidesencompassed herein include: HLA-A2: C A C T P E R M A and C T P E R M AE A; HLA-A3: F L E G C A C T P; HLA-B7/HLA-B35/HLA-B51: W P F L E G C AC T (Phoshorylated Thr residue marked in bold).

A significant feature of the peptide of the invention is its capabilityto recognise or elicit INF-γ-producing responder T cells, i.e. cytotoxicT cells (CTLs) that specifically recognise the particular peptide in aPBL population or tumour cells of a cancer patient (target cells). Thisactivity is readily determined by subjecting PBLs or tumour cells from apatient to an ELISPOT assay as described in reference (16) and in thefollowing examples. Prior to the assay, it may be advantageous tostimulate the PBL population or the tumour cells to be assayed bycontacting the cells with the peptide to be tested. Preferably, thepeptide Is capable of eliciting or recognising INF-γ-producing T cellsat a frequency of at least 1 per 10⁴ PBLs as determined by an ELISPOTassay as used herein. More preferably the frequency is at least 5 per10⁴ PBLs, most preferably at least 10 per 10⁴ PBLs, such as at least 50or 100 per 10⁴ PBLs.

The ELISPOT assay represents a strong tool to monitor survivin peptidespecific T-cell responses. However, although it has been shown thatELISPOT reactivity in most cases correlates with the capacity of theCLLs to lyse target cells, the conclusive evidence for this notion canonly be given directly. Such direct evidence is provided herein, as itwas demonstrated (see Example 2) that survivin reactive cells isolatedby means of HLA/peptide complexes possess the functional capacity oflysing target cells. Additionally, it was demonstrated that the isolatedCTLs specifically recognising a peptide of the invention were capable oflysing HLA-matched tumour cells of different origin, e.g. melanomas andbreast cancer. This finding strongly suggests that cancer cells ingeneral process and present the same endogenous survivin peptide.Therefore, a major implication of the findings herein is that thepeptides of the invention are expressed and complexed with HLA moleculeson a variety of cancer cells of different histological origins. Thisrenders these cancer cells susceptible to destruction by CTLs andemphasizes the potential usefulness of survivin immunization to controlthe growth of different neoplasms. The presence of spontaneousCTL-responses in PBLs and tumour cells to HLA-restrictedsurvivin-derived peptide epitopes from patients suffering from threeunrelated cancer types, i.e., breast cancer, melanoma and CLL, furthersubstantiates the universal immunotherapeutic potential of this tumourantigen.

Accordingly, in another preferred embodiment the peptide of theinvention is capable of eliciting INF-γ-producing cells in a PBLpopulation of a patient having a cancer disease where survivin isexpressed including a haematopoietic malignancy including chroniclymphatic leukemia and chronic myeloid leukemia, melanoma, breastcancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreascancer and prostate cancer. Specifically, the peptide of the inventionis able to elicit an immune response in the form of T cell havingcytotoxic effect against survivin expressing cells of a cancer cellline, including a cell line selected from the breast cancer cell lineMCF-7 and the melanoma cell line FM3.

In addition to their capacity to elicit immune responses in PBLpopulations and cancer cell lines, it was demonstrated that the peptidesof the invention are also capable of eliciting cytolytic immuneresponses in situ, i.e. in solid tumour tissues. This was demonstratedby providing HLA-peptide complexes, e.g. being multimerised and beingprovided with a detectable label, and using such complexes forimmunohistochemistry stainings to detect in a tumour tissue CTLs thatare reactive with the epitope peptide of the invention. Accordingly, afurther significant feature of the peptide of the invention is that itis capable of in situ detection in a tumour tissue of CTLs that arereactive with the epitope peptide.

It is contemplated that the peptides of the invention, in addition totheir capacity to bind to HLA molecules resulting in the presentation ofcomplexes of HLA and peptides on cell surfaces, which complexes in turnact as epitopes or targets for cytolytic T cells, may elicit other typesof immune responses, such as B-cell responses resulting in theproduction of antibodies against the complexes and/or a Delayed TypeHypersensitivity (DTH) reaction. The latter type of immune response isdefined as a redness and palpable induration at the site of injection ofthe peptide of the invention.

It is well known, that the different HLA molecules are of differentprevalence in the major human populations. Accordingly, there is arequirement for identifying peptide epitopes restricted to several HLAclass I molecules to extend the patient cohort that can be treatedaccording to the methods of the present invention. The characterisationof multiple survivin epitopes with different HLA restriction elementsbroadens the clinical potential of this target antigen in two importantways: (i) It increases the number of patients eligible for immunotherapybased on survivin-derived peptides. The HLA-A2 antigen is expressed byaround 50% of the Caucasian and Asian populations, the HLA-A1 and HLA-A3antigens are both expressed by around 25% of Caucasians and 5% ofAsians, whereas the HLA-A11 antigen is expressed by around 15% ofCaucasians and 30% of Asians. Even though these numbers cannot be summedup due to co-expression, a combination of peptides restricted by amultiplicity of these would certainly encompass most cancer patients,(ii) The collective targeting of several restriction elements in eachpatient Is likely to decrease the risk of immune escape by HLA-alleleloss. Loss of a single HLA allele is a significant component of MHCalterations described for cancer cells, whereas total loss of Class Iexpression is a rather infrequent event. Thus, with the identificationof survivin epitopes restricted to different HLA alleles, it is nowpossible to target more than one HLA-molecule simultaneously in patientswith allelic overlap.

Accordingly, based on the disclosure of the present invention the personof skill in the art would be able to develop highly immunogenicmulti-epitope vaccines. Preferably, such vaccines should be designed soas to facilitate a simultaneous delivery of the best-suitedsurvivin-derived peptides optionally in combination with other suitablepeptides and/or adjuvants as described hereinafter.

Furthermore, as previously described, there has been an increased focuson eliciting tumor-specific T helper cell immunity, i.e., vaccinatingwith class II-MHC restricted epitopes despite the fact that tumorsgenerally do not express class II MHC. This Is based on the recentfinding that the induction and efficacy of the vaccine-inducedanti-tumor response in many cases requires the cooperation oftumor-specific CD4 positive T_(h) cells. Thus, an important factordriving the development of vaccines having a more complex composition isthe desire to target multiple tumor antigens e.g. by designing vaccinescomprising or encoding a collection of carefully selected CTL and T_(h)cell epitopes.

Obviously, multi-epitope vaccines constitute an efficient way to raiseimmunity against epitopes derived from several different antigenswithout the need for introducing (genes encoding) potentially hazardousproteins such as oncoproteins. Such vaccines also permit selectiveinduction of immunity against subdominant and cryptic T cell epitopes,which can be especially important in the case of tumor-associatedautoantigens for which tolerance may exist for the epitopes that areprominently presented in normal tissues. Furthermore, antigen-presentingcells may fail to present certain epitopes that are expressed on tumorcells because of functional differences between the immunoproteasomes ofantigen-presenting cells and the ‘constitutive’ proteasomes present inmost tumor cells. In the case of peptide-based vaccines, such epitopescan be administered in an ‘MHC-ready’ form, which enables presentationthrough exogenous loading independently of antigen uptake and processingby host antigen-presenting cells.

It is evident that the findings of the present invention provide thebasis for therapeutic as well as diagnostic applications of thesurvivin-derived peptides.

Accordingly, in a further aspect the present invention provides apharmaceutical composition comprising one or more of the peptides of theinvention alone or in suitable combination with other proteins orpeptide fragments. In specific embodiments such other proteins orpeptide fragments include but are not limited to proteins involved inregulation of cell apoptosis or peptide fragments hereof. Suitableexamples of such proteins can be selected from the Bcl-2 protein family,e.g., the Bcl-2 protein, the Bcl-w protein, the Mcl-1 protein, theBcl-XL protein, and peptide fragments derived from any of the proteins.Other known apoptosis inhibitors include members of the inhibitor ofapoptosis protein (IAP) family such as X-IAP, C-IAP1 and C-IAP2 theseproteins are all relatively ubiquitously expressed whereas the inhibitorof apoptosis polypeptide ML-IAP has a rather selective expression, andis predominantly detected in melanomas. Thus, fragments of ML-IAPcapable of eliciting a specific T-cell response i.e a cytotoxic T-cellresponse or a helper T-cell response may optionally be included in thecomposition of the present invention.

Useful peptide fragments of ML-IAP include ML-IAP₂₄₅ (RLQEERTCKV) (SEQID NO:75), ML-IAP₂₈₀ (QLCPICRAPV) (SEQ ID NO:76), ML-IAP₉₀ (RLASFYDWPL)(SEQ ID NO:77), ML-IAP₁₅₄ (LLRSKGRDFV) (SEQ ID NO:78), ML-IAP₂₃₀(VLEPPGARDV) (SEQ ID NO:79), ML-IAP₉₈ (PLTAEVPPEL) (SEQ ID NO:80),ML-IAP₃₄ (SLGSPVLGL) (SEQ ID NO:81), ML-IAP₅₄ (QILGQLRPL) (SEQ IDNO:82), ML-IAP₉₉ (LTAEVPPEL) (SEQ ID NO:83), ML-IAP₈₃ (GMGSEELRL) (SEQID NO:84) and ML-IAP₂₈₈ (ELPTPRREV) (SEQ ID NO:85).

Additionally, the composition according to the present invention may beproved as a multiepitope vaccine comprising class I restricted epitopeand/or class II restricted epitopes as defined hereinbefore.

Example of a presently preferred multiepitope vaccines include “tailormade” combinations of survivin-derived peptide eptiopes depending of thetissue type of the given patient, e.g., a subject carrying HLA-A2,HLA-A3, and HLA-B35 phenotypes could be vaccinated with a vaccinecomprising sur1M2, sur9, sur18K10, sur46Y9, sur51Y9. Additionally, thepharmaceutical composition of the invention may advantageously compriseat least one further immunogenic protein or peptide fragment hereofselected from a protein or peptide fragment not belonging to or derivedfrom the survivin protein. In specific embodiments, the immunogenicprotein or peptide fragment thereof is derived from the Bcl-2 proteinfamily as described hereinbefore. A further immunogenic Bcl-2-derivedpeptide is an HLA-A2 restricted peptide having a sequence selected fromthe following: Bcl₁₇₂, Bcl₁₈₀, Bcl₂₀₈, and Bcl₂₁₄

As the peptides of the invention are relatively small molecules it maybe required in such compositions to combine the peptides with variousmaterials such as adjuvants, to produce vaccines, immunogeniccompositions, etc. Adjuvants, broadly defined, are substances whichpromote immune responses. Frequently, the adjuvant of choice is Freund'scomplete or incomplete adjuvant, or killed B. pertussis organisms, usede.g. in combination with alum precipitated antigen. A general discussionof adjuvants is provided in Goding, Monoclonal Antibodies: Principles &Practice (2nd edition, 1986) at pages 61-63. Goding notes, however, thatwhen the antigen of interest is of low molecular weight, or is poorlyimmunogenic, coupling to an immunogenic carrier is recommended. Examplesof such carrier molecules include keyhole limpet haemocyanin, bovineserum albumin, ovalbumin and fowl immunoglobulin. Various saponinextracts have also been suggested to be useful as adjuvants inimmunogenic compositions. Recently, it has been proposed to usegranulocyte-macrophage colony stimulating factor (GM-CSF), a well knowncytokine, as an adjuvant (WO 97/28816).

Accordingly, the invention encompasses a therapeutic composition furthercomprising any adjuvant substance including any of the above orcombinations thereof. It is also contemplated that the antigen, i.e. thepeptide of the invention and the adjuvant can be administered separatelyin any appropriate sequence.

The choice of antigen in the pharmaceutical composition of the inventionwill depend on parameters determinable by the person of skill in theart. As it has been mentioned, each of the different peptides of theinvention is presented on the cell surfaces by a particular HLAmolecule. As such, if a subject to be treated is typed with respect toHLA phenotype, a peptide/peptides are selected that is/are known to bindto that particular HLA molecule.

Alternatively, the antigen of interest is selected based on theprevalence of the various HLA phenotypes in a given population. As anexample, HLA-A2 is the most prevalent phenotype in the Caucasianpopulation, and therefore, a composition containing a survivin-derivedpeptide binding to HLA-A2 will be active in a large proportion of thatpopulation. However, the composition of the invention may also contain acombination of two or more survivin-derived peptides, each interactingspecifically with a different HLA molecule so as to cover a largerproportion of the target population. Thus, as examples, thepharmaceutical composition may contain a combination of a peptiderestricted to a HLA-A molecule and a peptide restricted to a HLA-Bmolecule, e.g. including those HLA-A and HLA-B molecules that correspondto the prevalence of HLA phenotypes in the target population, such ase.g. HLA-A2 and HLA-B35. Additionally, the composition may comprise apeptide restricted to an HLA-C molecule.

It is comtemplated that useful immunogenic compositions of theinventions in addition to a survivin-derived peptide as defined hereinmay comprise an immunologically effective amount of the survivin proteinas such as it is defined herein or an immunogenic fragment hereof.

The amount of the immunogenic peptide of the invention in thepharmaceutical composition may vary, depending on the particularapplication. However, a single dose of the immunogen is preferablyanywhere from about 10 μg to about 5000 μg, more preferably from about50 μg to about 2500 μg such as about 100 μg to about 1000 μg. Modes ofadministration include intradermal, subcutaneous and intravenousadministration, implantation in the form of a time release formulation,etc. Any and all forms of administration known to the art areencompassed herein. Also any and all conventional dosage forms that areknown in the art to be appropriate for formulating injectableimmunogenic peptide composition are encompassed, such as lyophilisedforms and solutions, suspensions or emulsion forms containing, ifrequired, conventional pharmaceutically acceptable carriers, diluents,preservatives, adjuvants, buffer components, etc.

The immunoprotective effect of the composition of the invention can bedetermined using several approaches. Examples hereof are provided in thefollowing examples. A further example on how to determine a CTL responseprovoked by the immunogenic composition is provided in WO 97/28816,supra. A successful immune response may also be determined by theoccurrence of DTH reactions after immunisation and/or the detection ofantibodies specifically recognising the peptide(s) of the vaccinecomposition.

In preferred embodiments, the pharmaceutical composition of theinvention is an immunogenic composition or vaccine capable of elicitingan immune response to a cancer disease. As used herein, the expression“immunogenic composition or vaccine” refers to a composition elicitingat least one type of immune response directed against cancer cells.Thus, such an immune response may be any of the types mentioned above: ACTL response where CTLs are generated that are capable of recognisingthe HLA/peptide complex presented on cell surfaces resulting in celllysis, i.e. the vaccine elicits the production in the vaccinated subjectof effector T-cells having a cytotoxic effect against the cancer cells;a B-cell response giving rise to the production of anti-cancerantibodies; and/or a DTH type of immune response.

In useful embodiments an immunogenic response directed against a cancerdisease is elicited by administering the peptide of the invention eitherby loading MHC class I molecules on antigen presenting cells (APCs) fromthe patient, by isolating PBLs from the patient and incubating the cellswith the peptide prior to injecting the cells back into the patient orby isolating precursor APCs from the patient and differentiating thecells into professional APCs using cytokines and antigen beforeinjecting the cells back into the patient. Thus, in one embodiment ofthe present invention, a method for treating cancer patients is onewherein the peptide is administered by presenting the peptide to thepatient's antigen presenting cells (APCs) ex vivo followed by injectingthe thus treated APCs back into the patient. There are at least twoalternative ways of performing this. One alternative is to isolate APCsfrom the cancer patient and incubate (load) the MHC class I moleculeswith the peptide. Loading the MHC class I molecules means incubating theAPCs with the peptide so that the APCs with MHC class I moleculesspecific for the peptide will bind the peptide and therefore be able topresent it to T cells. Subsequently, the APCs are re-injected into thepatient. Another alternative way relies on the recent discoveries madein the field of dendritic cell biology. In this case, monocytes (beingdendritic cell precursors) are isolated from the patient anddifferentiated in vitro into professional APC (or dendritic cells) byuse of cytokines and antigen. This is described in Examples 3 and 5,where adherent PBLs (being mainly monocytes) are cultured in vitrotogether with GM-CSF, IL-4 and TNF-α. Subsequently, the in vitrogenerated DCs are pulsed with the peptide and injected into the patient.

Due to the fact that survivin appears to be expressed in most cancerforms, it is very likely that vaccines of the invention can be providedto control any type of cancer disease where survivin is expressed. Thus,as examples, the vaccine composition of the invention is immunologicallyactive against a haematopoietic malignancy including chronic lymphaticleukemia and chronic myeloid leukemia, melanoma, breast cancer, cervixcancer, ovary cancer, lung cancer, colon cancer, pancreas cancer andprostate cancer.

From the above description, the skilled person will readily realise thatthe peptides of the invention are useful as cancer diagnostic tools,particularly so, as the peptides are derived from survivin expressed inall cancer types. Therefore, the peptides of the invention provide thebasis for developing universally applicable diagnostic and prognosticprocedures in respect of cancer diseases. Thus, in other usefulembodiments the composition of the invention is a composition for exvivo or in situ diagnosis of the presence in a cancer patient, e.g.based on the detection of survivin reactive T-cells among PBLs or intumour tissue.

Accordingly, there is, in still further aspects, provided a diagnostickit for ex vivo or in situ diagnosis of the presence of survivinreactive T-cells among PBLs or in tumor tissue comprising one or morepeptides of the invention, and a method of detecting in a cancer patientthe presence of survivin reactive T-cells, the method comprisingcontacting a tumor tissue or a blood sample with a complex of a peptideof the invention and a Class I HLA molecule or a fragment of suchmolecule and detecting binding of the complex to the tissue or the bloodcells.

Another useful diagnostic or prognostic approach is based on generatingantibodies in a heterologous animal species, e.g. murine antibodiesdirected against a human survivin-derived peptide of the invention,which can then be used, e.g. to diagnose for the presence of cancercells presenting the peptide. For such Immunisation purposes, the amountof peptide may be less than that used in the course of in vivo therapy,such as that mentioned above. In general, a preferred dose can rangefrom about 1 μg to about 750 μg of peptide. It is also possible toproduce monoclonal antibodies based on immunisation with a peptide ofthe invention. Accordingly, the present invention also relates to amolecule, in particular a monoclonal or polyclonal antibody including afragment hereof, that is capable of binding specifically to a peptide ofthe invention and to a molecule that is capable of blocking such abinding, e.g. an antibody raised against the monoclonal or polyclonalantibody directed against a peptide of the invention.

In one aspect, the invention provides a complex of a peptide of theinvention and a Class I HLA molecule or a fragment of such molecule,which is useful as a diagnostic reagent such as it is described supra.The complex is made by any conventional means including those describedin the following examples. Such a complex may be monomeric ormultimeric.

The present invention provides the means for alleviating or curing acancer disease. Accordingly, it is a further aspect of the invention touse the peptides as defined hereinbefore for the preparation of amedicament for the treatment of cancer. A still further aspect of thepresent invention relates to the use of a molecule or a composition asdefined hereinbefore for the preparation of a medicament for thetreatment of cancer. Preferably, a cancer disease associated with theexpression of survivin, including as examples: a haematopoieticmalignancy including chronic lymphatic leukemia and chronic myeloidleukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lungcancer, colon cancer, pancreas cancer and prostate cancer. The usecomprises administering to a patient suffering from the disease aneffective amount of the pharmaceutical composition according to theinvention, a molecule that is capable of binding specifically to apeptide of the invention and/or a molecule that is capable of blockingthe binding of such a molecule.

In some cases it will be appropriate to combine the use of the inventionwith a conventional cancer treatment such as radiotherapy orchemotherapy.

The invention will now be described in further details in the below,non-limiting examples and the figures, wherein

FIG. 1 illustrates T-cell response as measured in an ELISPOT in patientCLL1 to no peptide, Sur1 (LTLGEFLKL, SEQ ID NO:10) peptide and Sur9(ELTLGEFLKL, SEQ ID NO:3) peptide. PBLs were stimulated once withpeptide before plated at 6×10⁵ cells per well in duplicate. The averagenumber of spots per peptide was calculated using a CCD scanning deviceand a computer system,

FIG. 2 illustrates T-cell response as measured in an ELISPOT in patientCLL1 to no peptide, the peptide analogue Sur1L2 (LLLGEFLKL, SEQ IDNO:4), and the peptide analogue Sur1M2 (LMLGEFLKL, SEQ ID NO:5). PBLswere stimulated once with peptide before plated at 10⁴ cells per well induplicate. The average number of spots per peptide was calculated usinga CCD scanning device and a computer system,

FIG. 3 shows responses as measured in an ELISPOT in patient CLL2 andCLL3 to no peptide (black bar), the Sur1 (LTLGEFLKL, SEQ ID NO:10)peptide (grey bar), the Sur9 (ELTLGEFLKL, SEQ ID NO:3) peptide (whitebar), the analogue peptide Sur1L2 (LLLGEFLKL, SEQ ID NO:4) (light greybar), and the analogue peptide Sur1M2 (LMLGEFLKL, SEQ ID NO:5) (darkgrey bar). Each experiment was performed with 10⁵ cells per well induplicate, and the average number of spots was calculated,

FIG. 4 represents T cells that were Isolated from tumour infiltratedlymph nodes from patient Mel1, Mel2, and Mel3, stimulated once in vitroand analyzed in an ELISPOT assay for response to no peptide (black bar)the peptides Sur1 (LTLGEFLKL, SEQ ID NO:10) (grey bar) and Sur9(ELTLGEFLKL, SEQ ID NO:3) (white bar). Each experiment was performed induplicate with 10⁵ cells per well. In each experiment two wells withoutaddition of peptide was also included. The average number of spots perpeptide was calculated for each patient,

FIG. 5 shows functional activity of survivin specific CTLs. CTLs wereisolated from a melanoma infiltrated lymph node using survivin coatedmagnetic beads. (A) Specific lysis of melanoma cell lines; the HLA-A2positive FM3 (triangle) and the HLA-A2 negative FM45 (square). (B)Specific lysis of breast cancer cell lines; the HLA-A2 positive MCF-7(triangle) and the HLA-A2 negative BT-20 (square),

FIG. 6 shows frequency of survivin reactive CTLs in PBL from breastcancer patients. Reactivity was examined in three breast cancer patients(top, middle, and bottom, respectively) by the ELISPOT. For each patientthe assays were performed in the absence of peptide, in the presence ofsur1 peptide, in the presence of sur9, and in the presence of themodified sur1M2 peptide. 1×10⁴ effector cells per well were used. Thegraph depicts the quantification of reactive cells; grey columnsrepresent the average number of IFN-γ producing cells,

FIG. 7 illustrates HLA-35 binding of survivin-derived peptides andanalysis of the peptide-mediated recovery of HLA-B35 molecules bysurvivin-derived peptides. Lysates of metabolically labeled T2-B35 cellswere incubated at 4° C. in the presence of 50, 5, 0.5, 0.05 and 0.005 mMof peptide. The recovery of HLA-B35 was analyzed in an assembly assayand quantified subsequent to IEF-gel electrophoresis, using ImageGaugephosphorimager software (FUJI photo film Co., LTD., Japan). The C₅₀value is the concentration of the peptide required for half-maximalbinding to HLA-B35,

FIG. 8 shows spontaneous T-cell responses observed in PBLs from cancerpatients. A) The number of IFNγ spot forming cells measured in ELISPOTassay without peptide (white bars), with sur51-59 (black bars) orsur46-54 (gray bars), among in vitro stimulated PBLs from patient CLL5(10⁵ cells/well), HEM12 (10⁵ cells/well), and HEM8 (5×10⁴ cells/well).B) The number of spot forming cells among 1.7×10⁵ PBLs from HEM12,cultured for 10 days with peptide-pulsed matured autologous dendriticcells. The columns represent the average of two measurements,

FIG. 9 demonstrates spontaneous T-cell responses against native andmodified survivin peptides in melanoma patients. A) The number of spotforming cells measured in ELISPOT assay against sur51-59 and sur51Y9from patient FM25 in PBLs (4×10³ cells/well) and TILs (7×10⁴ cells/well)as well as TILs from FM45 (10⁴ cells/well). B) The number of spotforming cells measured in ELISPOT assay against sur46 and sur46Y9measured in TILs from FM74 (5×10³ cells/well). The columns represent theaverage of two measurements with the non-specific IFNγ releasesubtracted,

FIG. 10 illustrates binding affinity of survivin-derived peptides toHLA-A1. Class I MHC heavy chain bands were quantified on aPhosphorimager. The mount of stabilized HLA-A1 heavy chain is directlyrelated to the binding affinity of the added peptide. Thepeptide-mediated recovery of HLA-A1 (arbitrary units) induced by 40, 4,0.4, 0.04 μM of Sur93-101 (line), Sur93T2 (square), Sur49-58 (circle) orInfluenza A, PB1 591-599 (triangle),

FIG. 11 shows spontaneous responses against HLA-A1 restricted peptides.Spontaneous T-cell responses against survivin-derived peptides asmeasured by ELISPOT assay. The average number of peptide specific IFNγspots formed in response to Sur92-101, Sur38Y9, Sur47Y10, and Sur93T2among 5×10⁴ in vitro stimulated PBL or TIL from melanoma patients. Thepeptide specific responses showed were observed among analyses of 6 PBLsamples and 3 TIL samples from melanoma (MeI) patients. Non-specificIFNγ spots are subtracted. Bars: range of duplicates,

FIG. 12 shows spontaneous responses against HLA-A11 restricted peptides.Spontaneous T-cell responses against survivin-derived peptides asmeasured by the ELISPOT assay. The average number of peptide specificIFNγ spots formed in response to Sur53-62 among 5×10⁴ in vitrostimulated PBL or TIL from cancer patients. The peptide specificresponses showed were observed among analyses of 5 melanoma (MeI)patients (5 PBL, 1 TIL) and 2 CLL (CLL) patients (PBL). Non-specificIFNγ spots are subtracted. Bars: range of duplicates,

FIG. 13 illustrates spontaneous responses against HLA-A3 restrictedpeptides. Spontaneous T-cell responses against survivin-derived peptidesas measured by the ELISPOT assay. The average number of peptide specificIFNγ spots formed in response to Sur18K10 among 5×10⁴ in vitrostimulated PBL or TIL from melanoma patients. The peptide specificresponses showed were observed among analyses of 23 PBL samples and 4TIL samples from melanoma (MeI) patients. Non-specific IFNγ spots aresubtracted. Bars: range of duplicates,

FIG. 14 illustrates spontaneous responses against HLA-A2 restrictedpeptides. Spontaneous T cell responses against survivin-derived peptidesas measured by the ELISPOT assay. The average number of peptide specificIFNγ spots formed in response to the 11mer peptide, Sur18-28 among 5×10⁴in vitro stimulated PBL from cancer patients. The peptide specificresponses showed were observed among analyses of 10 PBL samples from 2melanoma (MeI), 6 CLL (CLL), and 2 mamma carcinoma (MC) patients.Non-specific IFNγ spots are subtracted. Bars: range of duplicates,

FIG. 15 illustrates spontaneous T cell responses againstsurvivin-derived peptides as measured by the ELISPOT assay. The averagenumber of peptide specific IFNγ spots formed in response to sur6-14(LPPAWQPFL) among 10⁵ in vitro stimulated PBL from five melanomapatients (mel25, mel26, mel3, mel6, mel39), two CLL patients (CLL1,CLL54) and 2 breast cancer patients (breast11, breast 15). Non-specificIFNγ spots are subtracted,

FIG. 16 illustrates the laboratory values of stable detection of LDH,cholinesterase, creatinine, hemoglobin, leucocytes and thrombocytesfollowing vaccination therapy of four patients (▴ RW, ● KN, − WWE, ▪GB), and

FIG. 17 demonstrates kinetic analysis of immunity to survivin peptidesassessed by IFNγ ELISPOT. PBMCs were obtained before the first DCvaccination and three months thereafter. The numbers of IFNγspot-forming cells above background are depicted.

In the following table, amino acid sequences for peptides used hereinand their respective SEQ ID NOs are listed:

SEQ ID NO: DESIGNATION SEQUENCE 1 Sur6 FLKLDRERA 2 Sur8 TLPPAWQPFL 3Sur9 ELTLGEFLKL 4 Sur1L2 LLLGEFLKL 5 Sur1M2 LMLGEFLKL 6 Sur 46-54CPTENEPDL 7 Sur51-59 EPDLAQCFF 8 Sur46Y9 CPTENEPDY 9 sur51Y9 EPDLAQCFY10 Sur1 LTLGEFLKL 11 C1 ILKEPVHGV 12 Sur2 RAIEQLAAM 13 Sur3 KVRRAIEQL 14Sur4 STFKNWPFL 15 Sur5 SVKKQFEEL 16 Sur7 TAKKVRRAI 17 Sur10 ETAKKVRRAI18 Sur 6-14 LPPAWQPFL 19 Sur 11-19 QPFLKDHRI 20 Sur 34-43 TPERMAEAGF 21C24 YPLHEQHQM 22 Sur14-22 LKDHRISTF 23 Sur38-46 MAEAGFIHC 24 Sur93-101FEELTLGEF 25 Sur47-56 PTENEPDLAQ 26 Sur49-58 ENEPDLAQCF 27 Sur92-101QFEELTLGEF 28 C1 VSDGGPNLY 29 sur14Y9 LKDHRISTY 30 sur93Y9 FEELTLGEY 31sur92Y9 QFEELTLGEY 32 sur34Y9 TPERMAEAGY 33 sur49Y9 ENEPDLAQCY 34Sur92T2 QTEELTLGEF 35 Sur92S2 QSEELTLGEF 36 Sur93T2 FTELTLGEF 37 Sur93S2FSELTLGEF 38 Sur38Y9 MAEAGFIHY 39 Sur47Y10 PTENEPDLAY 40 Sur 5-13TLPPAWQPF 41 Sur 53-61 DLAQCFFCF 42 Sur 54-62 LAQCFFCFK 43 Sur 95-103ELTLGEFLK 44 Sur 112-120 KIAKETNNK 45 Sur 13-22 FLKDHRISTF 47 Sur 53-62DLAQCFFCFK 50 Sur 103-112 KLDRERAKNK 51 Sur 112-121 KIAKETNNKK 52Sur 113-122 IAKETNNKKK 53 C3 ILRGSVAHK 54 Sur5K9 TLPPAWQPK 55 Sur53K9DLAQCFFCK 56 Sur54L2 LLQCFFCFK 57 Sur13K9 FLKDHRISTK 58 Sur18K10RISTFKNWPK 59 Sur113L2 ILKETNNKKK 60 SurEx3-A3-1 TIRRKNLRK 61SurEx3-A3-2 PTIRRKNLRK 62 Sur2b-A3-1 RITREEHKK 63 C4 AVFDRKSDAK 64 C6QPRAPIRPI 65 C7 RPPIFIRRL 66 Sur4-14 PTLPPAWQPFL 67 Sur18-28 RISTFKNWPFL68 Sur54-64 LAQCFFCFKEL 69 Sur86-96 FLSVKKQFEEL 70 Sur88-98 SVKKQFEELTL71 Sur103-113 KLDRERAKNKI 72 Ebv, BMLF1 GLCTLVAML 73 Hiv, Pol ILKEPVHGV74 Influenza A, ILRGSVAHK nucleoprotein 265-273

Example 1 Identification of a Cytotoxic T-Lymphocyte Response to theApoptosis Inhibitor Protein Survivin in Cancer Patients

Summary

Using CTL epitopes derived from survivin, specific T-cell reactivityagainst such antigens in peripheral blood from chronic lymphaticleukemia (CLL) patients and in tumor-infiltrated lymph nodes frommelanoma patients by ELISPOT analysis have been studied. CTL responsesto survivin-derived peptide epitopes were detected in three out of sixmelanoma patients and in three out of four CLL patients. No T-cellreactivity was detected in PBL from six healthy controls. Thus,survivin-derived peptides may serve as important and widely applicabletargets for anti-cancer immunotherapeutic strategies.

Introduction

The survivin protein was scanned for the presence of HLA-A*0201 (HLA-A2)binding peptide motifs and after successful identification, the peptideswere used to test for specific T-cell reactivity in leukemia andmelanoma patients by ELISPOT assay. In both patient cohorts CTLresponses to two survivin-derived peptide epitopes were detected,whereas no T-cell reactivity could be detected in the healthy controls.These data suggest that survivin represent a widely expressed tumorantigen recognized by autologous T cells.

Materials and Methods

Patients and Normal Controls

Peripheral vein blood samples from 4 patients diagnosed with CLL(designated CLL1-4) and blood samples from 6 normal individuals werecollected into heparinised tubes. PBLs were isolated using LYMPHOPREP®(density gradient medium) separation and frozen in fetal calf serum(FCS) with 10% di-methylsulphoxide. Additionally, T lymphocytes fromtumor-infiltrated lymph nodes were obtained from 6 melanoma patients(designated mel1-6). Freshly resected lymph nodes were minced into smallfragments, crushed to release cells into culture and cryopreserved. PBLswere available from 4 of the melanoma patients. All individuals includedwere HLA-A2 positive as determined by FACS analysis using the HLA-A2specific antibody BB7.2. The antibody was purified from hybridomasupernatant. Patient samples were obtained from the State UniversityHospital, Herlev, Denmark. Informed consent was obtained from thepatients prior to any of these measures.

Survivin-Derived Peptides

All peptides were obtained from Research Genetics (Huntsville, Ala.,USA) and provided at >90% purity as verified by HPLC and MS analysis.The peptides used are listed in Table 1.

TABLE 1 Peptides examined in this study and their binding affinity to HLA-A2Name Protein^(a) Sequence SEQ ID NO: C₅₀ (μM)^(b) C1 HIV-1 pol₄₇₆₋₄₈₄ILKEPVHGV 11 0.7 Sur1 Survivin₉₆₋₁₀₄ LTLGEFLKL 10 >100 Sur2Survivin₁₃₃₋₁₄₁ RAIEQLAAM 12 Not binding Sur3 Survivin₁₃₀₋₁₃₈ KVRRAIEQL13 >100 Sur4 Survivin₂₀₋₂₈ STFKNWPFL 14 Not binding Sur5 Survivin₈₈₋₉₆SVKKQFEEL 15 Not binding Sur6 Survivin₁₀₁₋₁₀₉ FLKLDRERA 1 30 Sur7Survivin₁₂₇₋₁₃₅ TAKKVRRAI 16 Not binding Sur8 Survivin₅₋₁₄ TLPPAWQPFL 230 Sur9 Survivin₉₅₋₁₀₄ ELTLGEFLKL 3 10 Sur10 Survivin₁₂₆₋₁₃₅ ETAKKVRRAI17 Not binding Sur1L2 LLLGEFLKL 4 1 Sur1M2 LMLGEFLKL 5 1 ^(a)The valuerange listed in subscript indicates the position of the peptide in thesurvivin sequence as disclosed in U.S. Pat. No. 6,245,523 ^(b)The C₅₀value is the concentration of the peptide required for half maximalbinding to HLA-A2 determined as described belowAssembly Assay for Peptide Binding to Class I MHC Molecules

Assembly assays for binding of the synthetic peptides to class I MHCmolecules metabolically labeled with [35S]-methionine were carried outas described (12,13). The assembly assay is based on stabilization ofthe class I molecules after loading of peptide to the peptidetransporter deficient cell line T2. Subsequently, correctly foldedstable MHC heavy chains are immunoprecipitated usingconformation-dependent antibodies. After IEF electrophoresis, gels wereexposed to phospholmager screens, and peptide binding was quantifiedusing the IMAGEQUANT® Phosphorlmager program (image analysis software)(Molecular Dynamics, Sunnyvale, Calif.).

Antigen Stimulation of PBLs

To extend the sensitivity of the ELISPOT assay, PBLs were stimulatedonce in vitro prior to analysis (14,15). Fresh and previously frozenPBLs gave similar results in the ELISPOT assay. On day 0, PBLs orcrushed lymph node were thawed and plated in 2 ml/well at aconcentration of 2×106 cells in 24-well plates (Nunc, Denmark) in AIM Vmedium (Life Technologies, Roskilde, Denmark), 5% heat-inactivated humanserum and 2 mM of L-glutamine in the presence of 10 μM of peptide. Ineach experiment a well without peptide was included. Two days later 300IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) wasadded to the cultures. The cultured cells were tested for reactivity inthe ELISPOT assay on day 12.

ELISPOT Assay

The ELISPOT assay used to quantify peptide epitope-specificinterferon-γ-releasing effector cells was performed as in (16). Briefly,nitrocellulose bottomed 96-well plates (MULTISCREEN® MAIP N45 (96-wellassay plate), Millipore, Hedehusene, Denmark) were coated withanti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells werewashed, blocked by AIM V® medium (serum-free media), and cells wereadded in duplicates at different cell concentrations. Peptides were thenadded to each well and the plates were incubated overnight. On thefollowing day, medium was discarded and the wells were washed prior toaddition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech).The plates were incubated for 2 hours, washed and Avidin-enzymeconjugate (AP-Avidin, Calbiochem, Life Technologies) was added to eachwell. Plates were incubated at RT for 1 hour and the enzyme substrateNBT/BCIP® (nitro-blue tetrazolium and5-bromo-4-chloro-3′-indolyphosphate) (Gibco, Life Technologies) wasadded to each well and incubated at room temperature for 5-10 min. Thereaction was terminated by washing with tap water upon the emergence ofdark purple spots. The spots were counted using the ALPHA IMAGER SYSTEM®(imaging system) (Alpha Innotech, San Leandro, Calif. USA) and thepeptide specific CTL frequency could be calculated from the numbers ofspot-forming cells. The assays were all performed in duplicate for eachpeptide antigen.

Results

Binding of Survivin Derived Peptides to HLA A2

The amino acid sequence of the survivin protein was screened for themost probable HLA-A2 nona- and decamer peptide epitopes, using the mainHLA-A2 specific anchor residues (17). Ten survivin-derived peptides weresynthesized and examined for binding to HLA-A2. An epitope from HIV-1pol476-484 (ILKEPVHGV, SEQ ID NO:11) (Table 1) was used as a positivecontrol. The peptide concentration required for half maximal recoveringof class I MHC (C₅₀ value) was 0.7 μM for the positive control. Incomparison, the peptide designated Sur9 (ELTLGEFLKL, SEQ ID NO:3) boundat an affinity of C₅₀=10 μM. The peptides designated Sur6 (FLKLDRERA,SEQ ID NO:1) and Sur8 (TLPPAWQPFL, SEQ ID NO:2), respectively bound toHLA-A2 at C₅₀=30 μM, whereas Sur1 (LTLGEFLKL, SEQ ID NO:10) and Sur3(KVRRAIEQL, SEQ ID NO:13) bound weaker (C₅₀>100 μM). Five of thepeptides examined (Sur2, Sur4, Sur5, Sur7, and Sur10) did not bind toHLA-A2.

Since Sur1 is a weak HLA-A2 binder, two analogue peptides designatedSur1L2 and Sur1M2, respectively in which a better anchor residue(leucine or methionine) replaced the native threonine at position 2 weresynthesized. Both of these peptides bind with almost similar highaffinity to HLA-A2 as the positive control (C₅₀=1 μM).

CTL Response to Survivin in CLL Patients

PBLs from four HLA-A2 positive CLL patients were stimulated once invitro before examination in the ELISPOT assay. This procedure was chosento extend the sensitivity of the ELISPOT. All of the above 10survivin-derived peptides were included in the first line ofexperiments. Responses were detected to Sur1 and Sur9 and only data forthese peptides are given in the figures. FIG. 1 shows CTL reactivity toSur1 and Sur9 as determined in patient CLL1. Each spot represents apeptide reactive, INF-γ-producing cell. The average number of spots perpeptide was calculated using a CCD scanning device and a computersystem. Fifty-two Sur9 peptide specific spots (after subtraction ofspots without added peptide) per 6×10⁵ were detected in the CLL1 patient(FIG. 1). No response was detected to the weak HLA-A2 binding peptideSur1, however the patient responded strongly to the strong HLA-A2binding peptide analogue Sur1M2 (35 peptide specific spots per 10⁴cells) (FIG. 2). No response was detected to the other strong HLA-A2binding peptide analogue Sur1L2 in this patient (FIG. 2). Patient CLL2responded strongly to Sur9 (128 peptide specific spots per 10⁵ cells)and weakly to Sur1 (22 peptide specific spots per 10⁵ cells) (FIG. 3).The response to the Sur1L2 analogue was only slightly increased relativeto the natural epitope, whereas the patient responded similarly stronglyto the Sur1M2 peptide as to the decamer peptide Sur9. In patient CLL3 aweak response to Sur9 was observed (FIG. 3). No response to Sur1 or themodified Sur1 peptides were observed in the patient. No survivinresponses were detected in the last patient CLL4 (data not shown). PBLsfrom 6 healthy HLA-A2 positive controls were analyzed to investigatewhether a response to survivin could be detected in healthy individuals.No response was observed in any of the controls to any of thesurvivin-derived peptides.

CTL Response to Survivin in Melanoma Patients

T lymphocytes isolated from tumour infiltrated lymph nodes from HLA-A2positive melanoma patients were examined. The freshly resected lymphnode was minced into small fragments and crushed to release cells intoculture. Cells were stimulated once with peptide in vitro beforeexamination in the ELISPOT assay. Survivin specific T cells weredetested in three of the six patients analyzed. A strong Sur9 responsewas detected in patient Mel2 and Mel3. A weaker response to the Sur1peptide was also detected in these patients (FIG. 4). In Mel1 theresponse to the weakly binding peptide Sur1 was stronger than theresponse to the stronger HLA-A2 binder Sur9 (FIG. 4). No response wasdetected in the tumor-infiltrated lymph nodes from the last threemelanoma patients (Mel4-6). PBLs from two of the survivin reactingpatients, Mel1 and Mel2, and from two of the non-reacting patients, Mel4and Mel5, were examined. No response could be detected to either Sur9 orSur1 in PBLs from any of these patients (data not shown).

Example 2 Spontaneous Cytotoxic T-Cell Responses to Survivin-Derived MHCClass I-Restricted T-Cell Epitopes In Situ and Ex Vivo in CancerPatients

Summary

Spontaneous cytotoxic T-cell responses to survivin-derived MHC class Irestricted T-cell epitopes were demonstrated in situ as well as ex vivoin breast cancer, leukemia, and melanoma patients. Moreover, survivinreactive T cells isolated by magnetic beads coated with MHC/peptidecomplexes were cytotoxic to HLA-matched tumours of different tissuetypes. Being a universal tumor antigen, survivin may serve as a widelyapplicable target for anti-cancer immunotherapy.

Materials and Methods

Construction of HLA-Peptide Complexes for T-Cell Staining and T-CellSorting

A recognition site for enzymatic biotinylation using biotin proteinligase (BirA) in fusion with the 5′-end of the extracellular domains ofHLA A*0201 (residues 1-275) was expressed in E. coli BL21 (DE3). Therecombinant protein was purified by size (SEPHADEX G 25® (gelfiltration), Pharmacia) and ion exchange (MONO-Q®, Pharmacia)chromatography from inclusion bodies solubilised in 8 M urea. The HLAA*0201 was folded in vitro by dilution in the presence of the modifiedsurvivin peptide Sur1M2 (LMLGEFLKL, SEQ ID NO:5) or the MAA peptidegp100154-163, and subsequently biotinylated as described previously (35,36).

After gel filtration on a Pharmacia SEPHADEX G 25® (gel filtration)column to remove unbound biotin, the protein was multimerised withstreptavidin-FITC conjugated dextran molecules (kindly provided by L.Winther, DAKO, Denmark) to generate multivalent HLA-dextran compoundsfor immunohistochemistry. The HLA A*0201 construct was a kind gift ofDr. Mark M. Davis (Dept. of Microbiology and Immunology, StanfordUniversity, Palo Alto, Calif.). Cell separation was performed aspreviously described (37). Briefly, 5×106 streptavidin-conjugatedmagnetic beads (Dynal, Oslo, Norway) were washed twice in 200 μl coldPBS, 0.5 μg peptide/A*0201 monomers were added and the mixture incubatedfor 15 min. at room temperature. After two washes these beads were mixedwith PBLs at a ratio of 1:10 and subsequently incubated for 1 h followedby a precipitation of bead-bound cells in a magnetic field. Theprecipitation step was repeated once.

Immunohistochemistry Stainings

For staining with FITC-conjugated multimeric peptide/MHC complexes,tissue sections were dried overnight and subsequently fixed in coldacetone for 5 min. All incubation steps were performed at roomtemperature and in the dark: (i) 45 min. of the primary antibody (1:100diluted), (ii) Cy 3-conjugated goat anti-mouse (1:500 diluted; code115-165-100, Jackson ImmunoResearch, obtained from Dianova, Hamburg,Germany) for 45 min. and finally (iii) the multimers for 75 min. Betweeneach step the slides were washed two times for 10 min. in PBS/BSA 0.1%.The slides were mounted in VECTASHIELD® (antifade mounting medium) andkept in the refrigerator until observed under the confocal microscope.

Cytotoxicity Assay

Conventional [51Cr]-release assays for CTL-mediated cytotoxicity werecarried out as described in (13). Target cells were autologousEBV-transformed B-cell lines, the HLA-A2 positive breast cancer cellline MCF-7 (available at ATCC®), the HLA-A2 positive melanoma cell lineFM3 (38), the HLA-A2 negative breast cancer cell line BT-20 (availablefrom ATCC®) and the HLA-A2 negative melanoma cell line FM45 (38). Allcancer cell lines expressed survivin as examined by RT-PCR (data notshown).

ELISPOT Assay

The ELISPOT assay was used to quantify peptide epitope-specific IFN-γreleasing effector cells and has been described previously (39).Briefly, nitrocellulose bottomed 96-well plates (MULTISCREEN® (96-wellassay plate) MAIP N45, Millipore) were coated with an anti-IFN-γantibody (1-D1K, Mabtech, Sweden) and non-specific binding was blockedusing AIM V® medium (serum-free media) (GibcoBRL, Life TechnologiesInc., Gaithersburg, Md., USA). Lymphocytes were added at different cellconcentrations together with the specific peptides and T2 cells andincubated overnight at 37° C. Following two washes the biotinylateddetection antibody (7-B6-1-Biotin, Mabtech) was added. Specific bindingwas visualised using alkaline phosphatase-avidin together with therespective substrate (GibcoBRL). The reaction was terminated upon theappearance of dark purple spots, which were quantitated using the ALPHAIMAGER SYSTEM® (imaging system) (Alpha Innotech, San Leandro, Calif.,USA). The peptides used for the ELISPOT were Sur1, Sur9 and the Sur1analogue peptide Sur1M2 as described in Example 1.

Results

In Situ Staining of HLA-A2/Survivin Reactive T Cells

In Example 1 two survivin-derived peptide epitopes recognized by T cellsin leukemia and melanoma, i.e., Sur1 were identified. The weak bindingaffinity of Sur1 to HLA-A2 was improved substantially by replacingthreonine at position 2 with a better anchor residue (methionine;Sur1M2). This measure enabled the construction of stable HLA-A2/peptidecomplexes. These complexes were multimerised using dextran molecules,which were conjugated with streptavidin and FITC. MultimerisedMHC-complexes were used to stain acetone-fixed, frozen material. Using aconfocal laser microscope, Sur1M2/HLA-A*0201 reactive CTLs could readilybe detected in situ in the tumor microenvironment. We depicted suchcells in the primary tumor and the sentinel lymph node of a stage IIImelanoma patient as well as in a primary breast cancer lesion. To ensurethe specificity of the staining, a series of negative controls wascarried out. Neither the use of peptide/HLA-dextran multimers withpeptides derived from the melanoma differentiation antigen gp100 on thesame tumour, nor Sur1M2/HLA-dextran multimers in case of a tumour sampleobtained from an HLA-A2 negative donor resulted in a positive staining.

Isolated Survivin Reactive CTLs Lyse Tumour Cell Lines of DifferentOrigin

To characterise the functional capacity of survivin-reactive CTLs, thesecells were isolated by means of magnetic beads coated withHLA-A2/Sur1M2-complexes (36). A freshly resected melanoma infiltratedlymph node was minced into small fragments and crushed to release cellsInto culture. Cells were stimulated once with peptide in vitro prior toisolation. One day after isolation IL-2 was added, and on day 5 thecapacity of these cells to kill tumour cells was tested either byELISPOT or in standard 51Cr release assays. First, by means of ELISPOTanalysis it was possible to establish that CTLs isolated using themodified Sur1M2/HLA-A2-complex also responded to the native Sur1 peptide(data not shown). Second, the cytotoxicity of the survivin reactive CTLsagainst the HLA-A2 positive melanoma cell-line FM3 (FIG. 5 A) and theHLA-A2 positive breast-cancer cell line MCF-7 (FIG. 5 B) was tested. Theisolated T cells effectively lysed both HLA-A*0201 cell lines. Incontrast, no cytotoxicity was observed against the HLA-A2 negativemelanoma cell line FM45 (FIG. 5 A) or the HLA-A2 negative breast cancercell line BT-20 (FIG. 5 B).

Survivin Reactivity Measured in PBL by ELISPOT

The presence of survivin reactive T cells in PBLs from ten HLA-A2positive breast cancer patients was examined by the ELISPOT. Beforeanalysis, PBLs were stimulated once in vitro to extend the sensitivityof the assay. Reactivity to the following survivin peptides wasexamined: Sur1, Sur9 and Sur1M2. Survivin specific T cells were detectedin six out of the ten HLA-A2 positive breast cancer patients.Representative examples are given in FIG. 6. In PBLs from two patients aresponse against Sur1 and the modified analogue Sur1M2, but not againstSur9 (FIG. 6, top, middle) was detected, in three patients a response toSur9 was detected, but not to Sur1 or Sur1M2 (FIG. 6 bottom), and onepatient responded only to Sur1M2. In contrast, no survivin responseswere detected in PBLs from 20 healthy HLA-A2 positive donors. Similarly,PBLs from fourteen HLA-A2 positive melanoma patients were examined.Survivin responses were present in seven of these patients (Table 2).Two patients responded to the Sur9 peptide, three to the Sur1M2 peptide,one to both Sur1 and SurM2, and one to all three peptides. In Example 1,T-cell response to survivin in 3 chronic lymphatic leukemia (CLL)patients was tested (Table 2; CLL1, CLL2, CLL3). These studies wereextended using PBLs from three additional CLL patients. Notably, allpatients produced a T-cell response to at least one survivin epitope(Table 2; CLL5, CLL6, CLL7). In addition, PBLs from one patientsuffering from chronic myeloid leukemia (CML) was examined. In thispatient, a response to all three peptides was identified (data notshown). The data are summarized in Table 2.

TABLE 2 Patients with survivin peptide-specific T lymphocytes in PBLs asmeasured by ELISPOT Patient Sur1 Sur9 Sur1M2 Melanoma a) P4 — —  97 P11— — 112 P13 — —  71 P15 61 — 101 P17 — 172 — P39 — 127 — P64 112  70 128Breast cancer b) B1 122  — 208 B2 67 —  72 B3 — 54 — B4 — 45 — B5 — 19 —B6 — —  24 CLL c) CLL1 — 27 320 CLL2 — 39 — CLL3 23 127 122 CLL5 — 100124 CLL6 — 121 360 CLL7 68 132 174 a) Frequency of reactive cells per10⁴; 14 patients examined. b) Frequency of reactive cells per 10⁴; 10patients examined. c) Frequency of reactive cells per 10⁵; 7 patientsexamined.

Example 3 HLA-B35-Restricted Immune Responses to Survivin-DerivedPeptides in Cancer Patients

Summary

In this study, two survivin-derived epitopes, which are restricted toHLA-B35 were identified and characterized. Specific T-cell reactivityagainst both of these epitopes was present in the peripheral blood frompatients with different haematopoietic malignancies and melanoma.Substitutions of the C-terminal anchor residue improved the recognitionby tumor infiltrating lymphocytes from melanoma patients. Furthermore,spontaneous cytotoxic T-cell responses to survivin in situ in a primarymelanoma lesion was demonstrated. These epitopes extends theapplicability of future vaccine strategies based on survivin peptides inrelation to malignancies as well as the HLA profile of the patientsinvolved.

In Examples 1 and 2, HLA-A2 restricted survivin-derived T-cell epitopeswere studied. Since HLA-A2 is only expressed in about 30% of theCaucasian population (63), peptide epitopes restricted to other HLAclass I molecules need to be identified to extend the fraction ofpatients that could be treated. In this study, two novel T-cell epitopesfrom survivin restricted to HLA-B35, which is expressed in 9% of theCaucasian population (63), were identified, and spontaneous immuneresponses to these survivin peptides were detected in patients withdifferent haematopoietic malignancies and melanoma.

Materials and Methods

Patients

Peripheral vein blood samples from cancer patients were collected, PBLswere isolated using LYMPHOPREP® (density gradient medium) separation,HLA-typed (Department of Clinical Immunology, University Hospital,Copenhagen) and frozen in FCS with 10% DMSO. Ten HLA-B35 positivepatients were selected for further analysis. These patients sufferedfrom melanoma, CLL, follicular lymphoma (FL), diffuse large B-celllymphomas (DLBCL) and Multiple Myeloma (MM), respectively. At the timeblood samples were collected patients had not been medically treatedwithin the previous four months. Additionally, tumor-infiltratinglymphocytes (TIL) isolated from lymph nodes were collected from three ofthe melanoma patients and frozen in FCS with 10% DMSO.

Peptides

Seven synthetic survivin-derived peptides were used in this study:Sur6-14, Sur11-19, Sur34-43, Sur46-54, Sur51-59, Sur46Y9, Sur51Y9, andone EBV-derived peptide, EBNA3A 457-466 (63). All peptides were obtainedfrom Research Genetics (Huntsville, Ala.) and provided at >90% purity,as verified by HPLC and MC analyses. The peptides are listed in Table 3below.

TABLE 3  HLA-B35 binding of survivin-derived peptides NameProtein and position Sequence SEQ ID NO: C₅₀ (μM) Sur6-14 Survivin₆₋₁₄LPPAWQPFL 18 >100 Sur11-19 Survivin₁₁₋₁₉ QPFLKDHRI 19 Not bindingSur34-43 Survivin₃₄₋₄₃ TPERMAEAGF 20 >100 Sur46-54 Survivin₄₆₋₅₄CPTENEPDL 6 20 Sur51-59 Survivin₅₁₋₅₉ EPDLAQCFF 7 13 Sur46Y9Modified peptide CPTENEPDY 8 4 Sur51Y9 Modified peptide EPDLAQCFY 9 1.5C24 EBNA3A₄₅₈₋₄₆₆ YPLHEQHQM 21 0.8Assembly Assay for Peptide Binding to MHC Class I Molecules

The assembly assay described in Examples 1 and 2 was used to measurebinding affinity of the synthetic peptides to HLA-B35 moleculesmetabolically labeled with [S35]methionine. Briefly, the assay is basedon peptide-mediated stabilization of empty HLA molecules released, uponcell lysis, from the TAP deficient cell line T2, stably transfected withHLA-B35 (kindly provided by Dr J. Haurum, Symphogen ApS, Lyngby,Denmark). Stably folded HLA-molecules were immunoprecipitated using theconformation-dependent mAb W6/32. The HLA molecules were separated byIEF electrophoresis, gels were exposed to phosphorimager screens(Imaging plate, FUJI photo film Co., LTD., Japan), analyzed and theamount of correctly folded HLA molecules were quantified usingImageGauge phosphorimager software (FUJI photo film Co., LTD., Japan).

Antigen Stimulation of PBLs

To extend the sensitivity of the ELISPOT assay, lymphocytes werestimulated once in vitro with peptide prior to analysis (14, 15). PBLsor TILs were thawed and stimulated with 50 μM of the individual peptideepitopes in 96-well plates for 2 h at 26° C. (5×10⁵-10⁶ cells perpeptide), and pooled for further 10 days of culture at 37° C. in x-vivowith 5% human serum (HS), in 24 well plates (Nunc, Roskilde, Denmark),with 2×10⁶ cells per well. At the second day of incubation 40 μg/ml IL-2(Apodan A/S, Denmark) were added. At day 10, the cultured cells weretested for reactivity in the ELISPOT assay.

The ELISPOT Assay

The ELISPOT assay used to quantify peptide specific, IFN-γ releasingeffector cells in PBLs or TILs collected from cancer patients wasperformed as described in Example 1. Briefly, nitrocellulose-bottomed96-well plates (MULTISCREEN® (96-well assay plate) MAIP N45; Millipore,Hedehusene, Denmark) were coated with mAb against human IFN-γ, 7.5 μg/ml(1-D1K; Mabtech, Nacka, Sweden). Wells were washed and blocked in x-vivo(X-VIVO 15® BioWhittacker (chemically defined, serum-free media),Molecular Applications Aps, Denmark) and cells were added in duplicatesat different concentrations. For antigen presentation, 10⁴ T2-B35 cells,with and without 10 μM peptide, were added per well. Plates wereincubated overnight, the cells discarded, and wells washed prior toaddition of biotinylated secondary antibody (7-B6-1-Biotin; Mabtech).Plates were incubated 2 h at room temperature, washed andavidin-alkaline phosphatase conjugate was added (AP-Avidin; Calbiochem,Life Technologies, Inc.). After 1 h of incubation at room temperature,the enzyme substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (Code No. K0598, DakoCytomation Norden A/S) was added, anddark purple spots emerged in 3-7 min. The reaction was terminated bywashing with tap water. Spots were counted using the ALPHA IMAGERSYSTEM® (imaging system) (Alpha Innotech, San Leandro, Calif.), and thefrequency of peptide specific T cells were calculated from the number ofspot forming cells.

All assays were performed in duplicates for each peptide antigen, andlymphocytes cultured in the same well, were tested in equal cell numberswith and without peptide, to measure the number of peptide specificcells in the culture.

Maturation of Dendritic Cells (DCs)

Adherent cells were isolated from PBLs after 2 h of culture. These werecultured for 10 additional days in RPMI 1640 (GIBCO®, InvitrogenCorporation, UK) with 10% FCS. 800 ng/ml GM-CSF (PreproTech, London, UK)and 40 ng/ml IL-4 (PreproTech) were added every third day. At day 10,DCs were matured for 24 h by adding 50 ng/ml TNF-.alpha. (PreproTech).After maturation, DCs were released and pulsed with 20 μM peptide in thepresence of 3 μg/ml. β2-microglobulin for 2 h at 26° C.

Isolation of Peptide Specific T Cells

Antigen specific cells were isolated using sur51Y9/HLA-B35-coatedmagnetic beads as described in Example 2. Biotinylated monomers ofHLA-B35 with sur51Y9 (obtained from ProImmune, Oxford, UK) were coupledto streptavidin coated magnetic beads (DYNABEADS® M-280 (magneticbeads), Dynal A/S, Oslo, Norway) by incubating 2.5 μg monomers with5×10⁶ beads in 40 μl PBS for 20 min. at room temperature. The magneticcomplexes were washed three times in PBS, using a magnetic device (DynalA/S, Oslo, Norway) and subsequently mixed with PBLs at a ratio of 1:10in PBS with 5% BSA, and rotated very gently for 1 h. Antigen specificCD8+ T cells associating with the magnetic complexes were gently washedtwo or three times. Isolated cells were resuspended several times inx-vivo supplemented with 5% human serum and incubated for 2 h before themagnetic beads were released and removed from the cell suspension. Theisolated antigen specific CD8+ T cells were used in ELISPOT assay toanalyze the cross-reactivity between the native and modified peptide.

TCR Clonotype Mapping by Denaturing Gradient Gel Electrophoresis (DGGE)

DGGE clonotype mapping of the human TCR BV regions 1-24 has beendescribed in details (66). Briefly, RNA was isolated using thePURESCRIPT® Isolation Kit (RNA isolation kit) (Gentra Systems Inc. MN)and transcribed cDNA was amplified by PCR using primers for the variableregions of the TCR beta chains in conjunction with a common constantregion primer. The computer program MELT87 was used to ensure that theamplified DNA molecules were suited for DGGE analysis provided a 50 bpGC-rich sequence (GC-clamp) was attached to the 5′-end of the constantregion primer. DGGE analysis was done in 6% polyacrylamide gelscontaining a gradient of urea and formamide from 20% to 80%.Electrophoresis was performed at 160 V for 4.5 hours in 1×TAE buffer ata constant temperature of 54° C.

Immunohistochemistry Stainings

Multimerised peptide/HLA complexes were used to identify antigenspecific T cells in situ in tumor lesions of cancer patients using theprocedure described in Example 2. Biotinylated sur51Y9/HLA-B35 monomerwas supplied by Proimmune limited, Oxford, UK. The biotinylated monomersof sur51Y9/HLA-B35 were multimerised with streptavidin-FITC-conjugateddextran molecules (kindly provided by L. Winther, DAKO, Glostrup,Denmark) to generate multivalent HLA-dextran compounds forimmunohistochemistry. Tissue sections were dried overnight andsubsequently fixed in cold acetone for 5 min. All the incubation stepswere performed in the dark at room temperature: (a) 45 min of theprimary antibody (1:100 diluted) (b) Cy 3-conjugated goat-anti-mouseantibody (1:500 diluted; code 115-165-100; Jackson ImmunoResearch,obtained from Dianova, Hamburg, Germany) for 45 min; and finally (c) themultimers for 75 min. Between each step, the slides were washed twotimes for 10 min in PBS/BSA 0.1%. The slides were mounted inVECTASHIELD® (antifade mounting medium) and kept in the refrigeratoruntil observed under the confocal microscope (Leica).

Results

Identification of HLA-835 Binding Survivin-Derived Peptides

The amino acid sequence of survivin was screened for nonameric anddecameric peptides with anchor residues, according to thepeptide-binding motif of HLA-B35 (67). Five peptides were selectedcontaining proline as the N-terminal anchor in position 2 andphenylalanine, leucine, isoleucine or tyrosine as C-terminal anchorresidues (Table 3). Assembly assay revealed two peptides, sur51-59(EPDLAQCFF, SEQ ID NO:7) and sur46-54 (CPTENEPDL, SEQ ID NO:6) that wereable to stabilise HLA-B35 efficiently. Additionally, two peptides,sur34-43 (TPERMAEAGF, SEQ ID NO:20) and sur6-14 (LPPAWQPFL, SEQ IDNO:18) showed a weak stabilization, whereas the remaining peptide didnot stabilize HLA-B35 at all. The peptide concentration required forhalf maximal recovery of HLA-B35 (C50) was estimated at 13 μM forsur51-59 and 20 μM for sur46-54. In comparison, the positivecontrol-epitope C24 from EBNA3A458-466 (YPLHEQHQM, SEQ ID NO:21) had anestimated C₅₀ value of 0.8 μM.

To enhance the binding affinity of sur46-54 and sur51-59 the C-terminalamino acid was replaced with tyrosine, a better anchor residue (67). Therecovery of HLA-B35 mediated by the modified peptides was analyzed inthe assembly assay, and C₅₀ values were estimated at 1.5 μM for sur51Y9and 4 μM for sur46Y9 (FIG. 7).

Spontaneous Immune Responses Against Native Peptide Epitopes

Initially, five patients were analyzed for spontaneous immune responsesto the four native HLA-B35 binding peptides sur51-59, sur46-54, sur34-43and sur6-14. These five patients had different haematopoieticmalignancies: HEM8 and HEM18 suffered from MM, HEM12 from FL, HEM9 hadDLBCL, and CLL5 had CLL.

INF-γ ELISPOT assays were performed on PBLs after 10 days of in vitrostimulation to detect peptide precursor CTLs. Spontaneous immuneresponses were detected against two of the native HLA-B35 bindingpeptides, sur51-59 and sur46-54. Two patients, HEM12 and CLL5 showed aresponse to both sur51-59 and sur46-54, whereas HEM8 only showed aresponse to sur51-59 (FIGS. 8A and B). No response could be detected inthe two remaining patients, HEM9 and HEM18, and no response could bedetected to the poorly binding peptides sur34-46 and sur6-14 in anypatients.

An alternative approach to in vitro stimulation was used in patientHEM12, i.e. PBLs were co-cultured with matured autologous dendriticcells pulsed with sur51-59 to stimulate a CTL response in vitro. PBLsfrom this culture showed strong reactivity towards sur51-59 in ELISPOT(FIG. 8B).

Increased Recognition of Modified Peptides

As described above, peptide modifications to enhance the HLA-B35affinity resulted in a 5-10-fold higher affinity for HLA-B35 relative tothe native peptides. A group of five melanoma patients were analyzed forspontaneous immune responses to both the native and modified peptides bymeans of ELISPOT assay. PBL samples were analyzed after in vitrostimulation, whereas TIL samples were analyzed directly. Spontaneousimmune responses were observed in either PBLs or TILs from three of thefive patients. FM25 showed reactivity against sur51-59 and sur51Y9 inboth PBL and TIL samples (FIG. 9A). FM45 responded only to the modifiedpeptide sur51Y9, with a strong response detectable in TILs. No PBLs wereavailable from this patient (FIG. 9A). FM74 showed a strong response tosur46Y9 in TIL, but no response to the native peptide was detectable(FIG. 9B). A weak response to sur46Y9 was also observed in PBLs fromFM74 (data not shown).

Cross-Reactivity Between the Native and Modified Peptide

The high affinity of sur51Y9 to HLA-B35 enables the production of stablemonomers of HLA-B35 with sur51Y9. Having established the presence ofsurvivin reactive T lymphocytes in tumor infiltrated lymph nodes andPBLs from different cancer patients, magnetic beads were coated withsuch HLA-B35/Sur51Y9-complexes and these were used to isolate survivinpeptide reactive T lymphocytes from PBL from patient CLL5. This patientshowed a strong response to sur51-59. Beads were tightly bound to thecell surface of the specific cells, as visualized by microscopy (datanot shown), permitting precipitation of antigen specific cells by amagnetic field. The isolated sur51Y9 specific cells responded stronglyto sur51-59, (FIG. 9), whereas no response could be detected in theremaining PBLs (data not shown). The isolation was analyzed by theRT-PCR/DGGE based TCR clonotype mapping. This technique allows theanalysis for T-cell clonality in complex cell populations, even if onlysmall numbers of cells are available. These analyses showed that 8distinct clones were isolated (data not shown).

Antigen Specific T Cells Present In Situ in a Melanoma Lesions

Sur51Y9/HLA-B35 monomers were multimerised using dextran moleculesconjugated with streptavidin and FITC. Multimerised MHC-complexes wereused to stain acetone-fixed, frozen material using the proceduredescribed in Example 2. Antigen specific cells were visualized using aconfocal laser microscope. Sections of primary melanoma from threepatients were analyzed, and Sur51Y9/HLA-B35-reactive CTLs could readilybe detected in situ in the tumor microenvironment in one of thepatients. Co-staining with a mAb against granzyme B showed that thesesurvivin specific CTLs released granzyme B, exerting cytotoxic activity,HLA-B35 negative melanoma patients were used as controls (data notshown).

Example 4 Identification of Novel Survivin-Derived CTL Epitopes withDifferent HLA-A-Restriction Profiles

Summary

Novel HLA-A1-, HLA-A2-, HLA-A3- and HLA-A11-restricted survivin epitopeswere characterised on the basis of CTL responses in cancer patients.These epitopes significantly increase the number of patients eligiblefor immunotherapy based on survivin-derived peptides. Additionally, thecollective targeting of several restriction elements is likely todecrease the risk of immune escape by HLA-allele loss.

Materials and Methods

Patients

Patient samples were received from the University of Würzburg, Germanyand the University Hospital in Herlev, Denmark. Informed consent wasobtained from the patients prior to any of these measures. Tissue typingwas conducted at Department of Clinical Immunology, University Hospital,Copenhagen, Denmark. Peripheral blood lymphocytes (PBL) from cancerpatients with melanoma, mamma carcinoma, and chronic lymphocyticleukemia (CLL) were isolated using Lymphoprep separation and frozen infetal calf serum (FCS) with 10% dimethylsulphoxide. Furthermore, Tlymphocytes from primary lesions and from tumor infiltrated lymph nodesfrom melanoma patients were obtained. Freshly resected tumor tissue wasminced into small fragments, and crushed to release tumor-infiltratinglymphocytes (TIL) for cryopreservation.

Peptides

All peptides were purchased from Invitrogen (Carlsbad, Calif., USA) andprovided at >80% purity as verified by HPLC and MS analysis. Allpeptides used are listed in Table 4, Example below.

Cell Lines

The human T2 cell line is a TAP1 and TAP2 defective hybrid of theB-LCL.174 and the T-LCL CEM cells and thus only express low levels ofHLA class I molecules (HLA-A*0201 and HLA-B*5101) at the cell surface.T2 cells transfected with HLA-A*0301 were kindly provided by Dr AMcMicheael, IMM, John Radcliffe Hospital, Oxford. T2 cells transfectedwith HLA-A*1101 were kindly provided by Dr M Masucci, MTC, KarolinskaInstitute, Stockholm, Sweden. The BM36.1 cell line is also defective inTAP function and has a similar phenotype as T2 with low expression ofHLA class I (HLA-A*0101, HLA-B*3501) at the surface. The BM36.1 cellswere kindly provided by Dr A Ziegler, Humboldt University, Berlin,Germany.

Assembly Assay for Peptide Binding to MHC Class I Molecules

The binding affinity of synthetic peptides (Invitrogen, Carlsbad,Calif., USA) to HLA-A1, -A2, -A3, or -A11 molecules metabolicallylabeled with [³⁵S]-methionine was measured in the assembly assay, asdescribed previously (12). The assay is based on peptide-mediatedstabilization of empty HLA molecules released upon cell lysis, from theTAP-deficient cell lines. Stably folded HLA-molecules wereimmune-precipitated using the HLA class I-specific,conformation-dependent mAb W6/32, and separated by isoelectric focusing(IEF) gel electrophoresis. MHC heavy chain bands were quantified usingthe ImageGauge Phosphorimager program (FUJI photo film Co., Carrollton,Tex., USA). The intensity of the band is proportional to the amount ofpeptide-bound class I MHC complex recovered during the assay.Subsequently, the extent of stabilization of the HLA-molecule isdirectly related to the binding affinity of the added peptide. Thepeptide concentration used to analyze the recovery of the HLA-moleculeswas 40, 4, 0.4, 0.04 μM for HLA-A1 and HLA-A11, and 100, 10, 1, 0.1,0.01 μM for HLA-A2 and HLA-A3. The C₅₀ value was subsequently calculatedfor each peptide as the peptide concentration sufficient for halfmaximal stabilization.

Antigen Stimulation of PBL

To extend the sensitivity of the ELISPOT assay, PBL were stimulated oncein vitro prior to analysis. At day 0, PBL or crushed lymph nodes werethawed and plated as 2×10⁶ cells in 2 ml/well in 24-well plates (Nunc,Roskilde, Denmark) in x-vivo medium (Bio Whittaker, Walkersville, Md.),5% heat-inactivated human serum, and 2 mM of L-glutamine in the presenceof 10 μM of peptide. Two days later 20 IU/ml recombinant interleukin-2(IL-2) (Chiron, Ratingen, Germany) was added to the cultures. Thecultured cells were tested for reactivity in the ELISPOT on day 10.

ELISPOT Assay

The ELISPOT assay was used to quantify peptide epitope-specificinterferon-.gamma. releasing effector cells as previously described(16). Briefly, nitrocellulose bottomed 96-well plates (MULTISCREEN® MAIPN45 (96-well assay plate), Millipore, Hedehusene, Denmark) were coatedwith anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells werewashed, blocked by X-vivo medium, and the cells were added in duplicatesat different cell concentrations. The peptides were then added to eachwell and the plates were incubated overnight. The following day, mediawas discarded and the wells were washed prior to addition ofbiotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plateswere incubated for 2 hours, washed, and avidin-alkaline phosphataseconjugate (Calbiochem, Life Technologies, Inc. San Diego, Calif., USA)was added to each well. The plates were incubated at room temperaturefor one hour, washed, and the enzyme substrate NBT/BCIP® (nitro-bluetetrazolium and 5-bromo-4-chloro-3′-indolyphosphate) (DakoCytomationNorden A/S, Glostrup, Denmark) was added to each well and incubated atRT for 5-10 min. Upon the emergence of dark purple spots, the reactionwas terminated by washing with tap-water. The spots were counted usingthe IMMUNOSPOT® Series 2.0 Analyzer (plate reader) (CTL Analyzers, LLC,Cleveland, US) and the peptide specific CTL frequency could becalculated from the numbers of spot-forming cells. All assays wereperformed in duplicates for each peptide antigen.

Results

Identification of HLA-A1 Restricted Survivin Epitopes

Binding of Survivin-Derived Peptides to HLA A1

The amino acid sequence of the survivin protein was screened for themost probable HLA-A1 nonamer or deca-mer peptide epitopes, using themain HLA-A1 anchor residues, aspartic acid (D), glutamic acid (E) atposition 3 and tyrosine (Y), phenylalanine (F) at the C-terminus.Accordingly, six survivin-derived peptides were synthesized and examinedfor binding to HLA-A1 (table 4). Additionally, the two peptides Sur38-46(MAEAGF1HC)(SEQ ID NO:23) and Sur47-56 (PTENEPDLAQ) (SEQ ID NO:25) wasincluded, in spite they only contain one of the main anchors, since bothwere identified as possible good binders by the predictive algorithm byRammensee et al. C₅₀ values were estimated for each peptide as thepeptide concentration needed for half maximal stabilization of HLA-A1(table 4). However, only one of these peptides Sur92-101 (QFEELTLGEF)(SEQ ID NO: 27) bound with almost similar high affinity as a knownpositive control epitope from the Influenza A protein, basic polymerase1 (PB1) (VSDGGPNLY) as exemplified in FIG. 10. Sur93-101 (FEELTLGEF)(SEQ ID NO:24) had a low binding affinity for HLA-A1, whereas none ofthe other peptides analyzed bound to HLA-A1 (Table 4). Consequently, wesynthesized a number of analogue peptides in which better anchorresidues replaced the natural amino acids. We modified the two peptidesSur38-46 (MAEAGF1HC) (SEQ ID NO:23) and Sur47-56 (PTENEPDLAQ) (SEQ IDNO:25) introducing tyrosine (Y) instead of cysteine (C) or glutamine (Q)respectively at the C-terminus. Both of the modified peptides boundstrongly to HLA-A1 (table 4). Additionally, we substituted the aminoacids at position 2 with the auxiliary anchors threonine (T) or serine(S) in the two peptides Sur92-101 and Sur93-101. These modifications didnot have a positive effect of the binding of Sur92-101 to HLA-A1. Incontrast, the Sur93T2 (FTELTLGEF) (SEQ ID NO:36) bound with highaffinity to HLA-A1 (Table 4). FIG. 10 illustrates the binding of thenative low affinity peptide Sur93-101, the high affinity modifiedpeptide Sur93T2 and the non-binding peptide Sur49-58 as compared to thepositive control epitope from influenza. Finally, we modified Sur14-22,Sur34-43, Sur49-58, Sur51-59, Sur92-101, and Sur93-101 with tyrosine (Y)at the C-terminus, however this did not improve binding affinity toHLA-A1 for any of these peptides (data not shown).

HLA-A1 Restricted CTL Responses Against Survivin-Derived Peptides inCancer Patients

PBL from six melanoma patients and TIL from three melanoma patients wereanalyzed for the presence of CTL specific against any of the four highaffinity survivin deduced peptides Sur38Y9, Sur47Y10, Sur92-101, andSur93T2 by means of ELISPOT. T-cell reactivity against at least one ofthe survivin-derived peptides was observed in three PBL samples and oneTIL sample from the total of nine patients analyzed. As seen in FIG. 11,PBL from one patient, Mel.A1-3 hosted a T-cell response against all fourpeptides, Sur38Y9, Sur47Y10, Sur92-101, and Sur93T2. Mel.A1-2 showedresponses against Sur47Y10, Sur92-101 and Sur93T2, whereas inMel.A1-1/TIL and Mel.A1-4/PBL responses were observed against Sur47Y10and Sur93T2, respectively (FIG. 11).

In addition, ten melanoma patients were tested for immune reactivityagainst the native peptides Sur93-101, Sur38-46 and Sur47-56 by means ofELISPOT; however, no peptide-specific responses were detected in any ofthese patients (data not shown).

Identification of HLA-A11 Restricted Survivin Epitopes

Binding of Survivin-Derived Peptides to HLA-A11

The amino acid sequence of the survivin protein was screened for nonameror deca-mer peptides with binding motifs corresponding to that of theHLA-A3 super-family, including HLA-A3 and HLA-A11. Peptide sequenceswith the main anchor residues, leucine (L) in position 2 and lysine (K)at the C-terminus, were chosen together with peptide sequences havingrelated amino acids at these positions according to the predictivealgorithm by Rammensee et al. (table 4).

Thirteen peptides were predicted from the protein sequence of survivinand analyzed for binding to HLA-A11 and HLA-A3. Three of these peptides,Sur53-62 (DLAQCFFCFK) (SEQ ID NO:47), Sur54-62 (LAQCFFCFK) (SEQ IDNO:42) and Sur112-120 (KIAKETNNK) (SEQ ID NO:44) bound HLA-A11 with highaffinity, comparable to the viral epitope from EBV nuclear antigen 4(AVFDRKSDAK) (SEQ ID NO:63). In addition, one peptide, Sur112-121(KIAKETNNKK) (SEQ ID NO:51) bound weakly to HLA-A11 (Table 4).

HLA-A11 Restricted CTL Responses Against Survivin-Derived Peptides inCancer Patients

PBL from five melanoma patients and two CLL patients were tested forT-cell reactivity against the four HLA-A11 binding peptides Sur53-62;Sur54-62, Sur112-120, and Sur112-121. We were able to detect responsesagainst the survivin-derived peptide Sur53-62 in PBL from two of themelanoma patients, Mel.A11-1, Mel.A11-2, by means of ELISPOT (FIG. 12).Additionally, we were able to detect Sur53-62 specific T-cells amongtumor infiltrating lymphocytes (TIL) from a tumor infiltrated lymph nodein patient Mel.A11-2 (FIG. 12). In the patient Mel.A11-1 a strong immuneresponse against the survivin peptide Sur53-62 was observed in fivedifferent blood samples taken over a period of two years (data notshown).

Identification of HLA-A3 Restricted Survivin Epitopes

Binding of Survivin-Derived Peptides to HLA-A3

The survivin-derived peptides predicted for binding to the HLA-A3super-family were additionally analyzed for the binding to HLA-A3. Onlytwo of the peptides Sur112-120 (KIAKETNNK) (SEQ ID NO:44) and Sur112-121(KIAKETNNKK) (SEQ ID NO:57) bound HLA-A3 with high affinity, similar tothe viral epitope, Influenza A nucleoprotein 265-273 (ILRGSVAHK) (SEQ IDNO:74) (Table 4). Furthermore, two peptides Sur53-62 (DLAQCFFCFK) (SEQID NO:47) and Sur95-103 (ELTLGEFLK) (SEQ ID NO:43) bound weakly toHLA-A3.

Some of the peptides with no detectable binding were modified in anattempt to increase the binding affinity for HLA-A3. Thus, wesynthesized two analogue peptides of Sur54-62 and Sur113-122 in which abetter anchor residue leucine (L) replaced the natural alanine (A) atposition 2. Sur54L2 (LLQCFFCFK) (SEQ ID NO:56) bound HLA-A3 with highaffinity, whereas Sur113L2 (ILKETNNKKK) (SEQ ID NO:59) only bound weakly(Table 4). In addition, we synthesized four analogue peptides ofSur5-13, Sur13-22, Sur18-27, and Sur53-61 in which the better anchorresidue lysine (K) replaced the natural phenylalanine (F) at theC-terminus. Sur5K9 (TLPPAWQPK) (SEQ ID NO:54) and Sur18K10 (RISTFKNWPK)(SEQ ID NO:58) bound to HLA-A3 with high affinity, whereas thesubstitutions had no detectable effect on the binding to HLA-A3 ofSur13K9 (FLKDHRISTK) (SEQ ID NO:57) and Sur53K9 (DLAQCFFCK) (SEQ IDNO:55) compared to the native analogues.

HLA-A3 Restricted CTL Responses Against Survivin-Derived Peptides inCancer Patients

Nine samples from melanoma patients (five PBL and four TIL) wereanalyzed for immune reactivity against the two native high affinityHLA-A3 binding peptides Sur112-120 and Sur112-121, as well as the twonative, weak binding peptides Sur53-62 and Sur95-103. However, no immuneresponses against these peptides could be detected by ELISPOT in any ofthe patients. Subsequently, the same patients were analyzed forspontaneous immune reactivity against the three high affinity, modifiedsurvivin-derived peptides, Sur5K9, Sur18K10, and Sur54L2. CTL reactivitywas detected against Sur18K10 in TIL samples from three patients,Mel.A3-1, Mel.A3-2, Mel.A3-3 (FIG. 13). No responses were detectedagainst the two other peptides, Sur5K9 and Sur54L2. To further verifythese responses, PBL from additional eighteen melanoma patients wereanalyzed for CTL reactivity against Sur18K10. Three responding patients,Mel.A3-4, Mel.A3-5, and Mel.A3-6, were found among these, resulting in atotal of six responding patients among the twenty-seven patientsanalyzed (FIG. 13).

Identification of a Novel HLA-A2 Restricted Survivin Epitope

Binding of 11-Mer Survivin-Derived Peptides to HLA-A2

The amino acid sequence of the survivin protein was screened for themost probable HLA-A2 11-mer peptide epitopes, using the main HLA-A2specific anchor residues. Six survivin deduced peptides were synthesizedand examined for binding to HLA-A2. None of the peptides examined boundwith similar high affinity as a known positive control epitope fromEpstein-Barr virus BMLF₂₈₀₋₂₈₈ peptide (GLCTLVAML) (SEQ ID NO:72) (Table4). The peptide concentration required for half maximal recovery ofHLA-A2 (C₅₀ value) was 0.9 μM for the positive control. In comparison,the peptides Sur18-28 (RISTFKNWPFL) (SEQ ID NO:67) and Sur86-96(FLSVKKQFEEL) (SEQ ID NO:69) bound weakly to HLA-A2 (C₅₀=69 μM and 72 μMrespectively). However, the two known HLA-A2-restricted survivinepitopes bound in a similar way weakly to HLA-A2; Sur95-104 (ELTLGEFLKL)(SEQ ID NO:43) bound with intermediate affinity (C₅₀=10 μM) whereasSur96-104 (LTLGEFLKL) (SEQ ID NO:10) bound only weakly (C₅₀>100 μM). Theremaining four 11-mer peptides examined (Sur-4-14 (PTLPPAWQPFL) (SEQ IDNO:66), Sur54-64 (LAQCFFCFKEL) (SEQ ID NO:68), Sur88-98 (SVKKQFEELTL)(SEQ ID NO:70), and Sur103-113 (KLDRERAKNKI) (SEQ ID NO:74)) did notbind to HLA-A2.

HLA-A2 Restricted CTL Responses Against Survivin-Derived Peptides inCancer Patients

PBL from ten cancer patients (two melanoma (Mel), six CLL (CLL), and twomamma carcinoma (MC) patients) was initially analyzed to investigatewhether the two weak binding 11mer peptides, Sur18-28 and Sur86-96 werepresented by HLA-A2 and recognized by the immune system of cancerpatients. CTL responses against Sur18-28 were found in PBL from two ofthe ten patients analyzed (CLL-1, CLL-2, FIG. 14), whereas no responsescould be detected against Sur86-96 (data not shown). To further verifythese Sur18-28 specific responses, PBL from additional twelve patients(seven melanoma, one CLL, and four mamma carcinoma patients) wereanalyzed for CTL reactivity against this peptide. Among these, fourpatients (CLL-3, MC-1, MC-2, Mel.A2-1) had Sur18-28 specific immuneactivity detectable by ELISPOT (FIG. 14). Thus, altogether PBL from sixout of twenty-two patients analyzed hosted a CTL response againstSur18-28.

Identification of HLA-B7 Restricted Survivin Epitopes

Binding of Survivin Derived Peptides to HLA-B7

The amino acid sequence of the survivin protein was screened forpeptides of nine to ten amino acids, with anchor residues according tothe peptide binding motif of HLA-B7. Five peptides were selected andanalyzed for their ability to stabilize HLA-B7 in the assembly assay.C₅₀ values were estimated for each peptide as the peptide concentrationneeded for half maximal stabilization of HLA-B7 (table 4). Twosurvivin-derived peptides, sur6-14 (LPPAWQPFL) (SEQ ID NO:18) andsur11-19 (QPFLKDHRI) (SEQ ID NO:19) stabilized HLA-B7 weakly, with C₅₀values above 100 μM; whereas sur46-54 (CPTENEPDL) (SEQ ID NO:6),sur51-59 (EPDLAQCFF) (SEQ ID NO:7), and sur34-43 (TPERMAEAGF) (SEQ IDNO:20) did not bind to HLA-B7 (table 4).

HLA-B7 Restricted CTL Responses Against Survivin Derived Peptides inCancer Patients

HLA-B7 positive PBL from five melanoma patients (mel25, mel26, mel3,mel6, mel39), two CLL patients (CLL1, CLL54) and 2 breast cancerpatients (breast11, breast 15) were tested for T-cell reactivity againstthe weak HLA-B7 binding peptides sur6-14 (LPPAWQPFL) (SEQ ID NO:18) andsur11-19 (QPFLKDHRI) (SEQ ID NO:19). We were able to detect a strongspontaneous CTL response against the survivin derived peptide sur6-14 inPBL in a CLL patient and in a breast cancer patient (FIG. 15).Additionally, we were able to detect a weak response against thispeptide in the melanoma patient mel3 (FIG. 15).

Summary of HLA Allele-Restricted Immune Responses to Survivin-DerivedPeptides in Cancer Patients

A range of survivin-derived peptides comprising 9-11 amino acid residueswere tested for binding to the following HLA alleles: HLA-A1, HLA-A3,HLA-A11 and HLA-B7 using the assembly assay for peptide binding to MHCclass I molecules described in the preceding examples. In addition,several of the peptides were tested for their capacity to elicit a CTLimmune response using the ELISPOT assay as also described above.

A summary of the results, including results obtained in the previousexamples, are given in the below Table 4:

TABLE 4  C₅₀ and ELISPOT data for selected survivin-derived peptides HLAPeptide SEQ ID allele length Position Sequence C₅₀ (μM) Remarks NO:Footnotes HLA-A1  9 mer Sur14-22 LKDHRISTF NB 22 Sur51-59 EPDLAQCFF NB 7Sur38-46 MAEAGFIHC NB 23 Sur93-101 FEELTLGEF >100 24 10 mer Sur34-43TPERMAEAGF NB 20 Sur47-56 PTENEPDLAQ NB 25 Sur49-58 ENEPDLAQCF NB 26Sur92-101 QFEELTLGEF 2 27 Control C1 VSDGGPNLY 0.8 28 peptide Modifiedsur14Y9 LKDHRISTY NB 29 peptides sur51Y9 EPDLAQCFY Weak 9 bindingsur93Y9 FEELTLGEY NB 30 sur92Y9 QFEELTLGEY NB 31 sur34Y9 TPERMAEAGY NB32 sur49Y9 ENEPDLAQCY NB 33 Sur92T2 QTEELTLGEF 2 34 Sur92S2 QSEELTLGEF100 35 Sur93T2 FTELTLGEF 1 36 Sur93S2 FSELTLGEF 30 37 Sur38Y9 MAEAGFIHY0.8 38 Sur47Y10 PTENEPDLAY 0.4 39 HLA-A2 Sur4-14 PTLPPAWQPFL NB 66Sur18-28 RISTFKNWPFL 69 67 Sur54-64 LAQCFFCFKEL NB 68 Sur86-96FLSVKKQFEEL 72 69 Sur88-98 SVKKQFEELTL NB 70 Sur103-113 KLDRERAKNKI NB71 Control EBV,BMLF1 GLCTLVAML 3 72 peptide HIV, Pol ILKEPVHGV 0.2 73HLA-A3  9 mer Sur 5-13 TLPPAWQPF NB 40 Sur 53-61 DLAQCFFCF NB 41Sur 54-62 LAQCFFCFK NB 42 Sur 95-103 ELTLGEFLK >100 43 Sur 112-120KIAKETNNK 2 44 i 10 mer Sur 13-22 FLKDHRISTF NB 45 Sur 18-27 RISTFKNWPFNB 46 Sur 53-62 DLAQCFFCFK 100 47 ii Sur 84-93 CAFLSVKKQF NB 48Sur 101-110 FLKLDRERAK NB 49 Sur 103-112 KLDRERAKNK NB 50 Sur 112-121KIAKETNNKK 1 51 Sur 113-122 IAKETNNKKK NB 52 Control C3 ILRGSVAHK0.1-0.3 53 peptide Modified Sur5K9 TLPPAWQPK 2 54 peptides Sur53K9DLAQCFFCK NB 55 Sur54L2 LLQCFFCFK 1 56 Sur13K9 FLKDHRISTK NB 57 Sur18K10RISTFKNWPK 0.02 58 Sur113L2 ILKETNNKKK >100 59 SurEx3-A3-1 TIRRKNLRK 0.560 iii SurEx3-A3-2 PTIRRKNLRK NB 61 Sur2b-A3-1 RITREEHKK NB 62 ControlInfluenza A, ILRGSVAHK 0.1 74 peptide nucleoprotein 265-273 HLA-A11 9 mer Sur 5-13 TLPPAWQPF NB 40 Sur 53-61 DLAQCFFCF NB 41 Sur 54-62LAQCFFCFK 0.4 42 Sur 95-103 ELTLGEFLK NB 43 Sur 112-120 KIAKETNNK 1 4410  mer Sur 13-22 FLKDHRISTF NB 45 Sur 18-27 RISTFKNWPF NB 46 Sur 53-62DLAQCFFCFK 5 47 Sur 84-93 CAFLSVKKQF NB 48 Sur 101-110 FLKLDRERAK NB 49Sur 103-112 KLDRERAKNK NB 50 Sur 112-121 KIAKETNNKK >100 51 ivSur 113-122 IAKETNNKKK NB 52 Control C4 AVFDRKSDAK 0.2 63 peptide HLA-B7 9 mer Sur 6-14 LPPAWQPFL >100 18 v Sur 11-19 QPFLKDHRI >100 19Sur 46-54 CPTENEPDL NB 6 Sur 51-59 EPDLAQCFF NB 7 10 mer Sur 34-43TPERMAEAGF NB 20 Control C6 QPRAPIRPI 0.1 64 peptides C7 RPPIFIRRL 0.565 i An response was observed against the peptide Sur112-120 in onelymphoma patient (HEM34) by means of ELISPOT. ii Responses were detectedagainst the peptide Sur53-62 in 3 lymphoma patients (HEM9, 11, 34) bymeans of ELISPOT. iv A weak response was observed in a melanoma patient(FM-TIL95) by means of ELISPOT. vii A response was observed againstSur112-121 in a melanoma patient (PM6), most evident in metastaticlymph-node suspension, and weaker in the TIL from primary tumor and PBLby means of ELISPOT. viii An response against the peptide Sur6-14 wasobserved in a CLL patient (CLL9), and a weaker response was observed ina lymphoma patient by means of elispot (HEM 21) (data not shown.

Example 5 Therapeutic Trial Procedures Using Survivin-Derived Peptidesas Immunogens

Summary

Five heavily pretreated stage IV melanoma patients were vaccinated withthe modified HLA-A2-restricted survivin epitope, namely the sur1M2peptide, presented by autologous dendritic cells in a compassionate usesetting. Four of the patients mounted strong T-cell response to thisepitope as measured by ELISPOT assay. Furthermore, in situpeptide/HLA-A2 multimer staining revealed the infiltration of survivinreactive cells into both visceral and soft tissue metastases. Notably,vaccination associated toxicity was not observed. The data demonstratethat it is feasible to induce T-cell response against survivin, even inlate stage melanoma patients, and that these vaccinations are welltolerated.

Materials and Methods

Patient Eligibility Criteria and Treatment Regimen

All clinical procedures were in accordance with the Declaration ofHelsinki and all patients provided informed consent prior to therapy.Stage IV cutaneous or uveal melanoma patients were eligible when theirdisease was progressive despite at least two different chemo-, immuno,or chemoimmunotherapies. In addition, a patients had to be 18 years orolder, express HLAA*0201, and suffer from measurable disease validatedby cranial, thoracic and abdominal computed tomography scans. Patients'Karnofsky index had to be 60% or better. No systemic chemo-, and/orimmunotherapy was allowed within 4 week prior to vaccination. Importantexclusion criteria were evidence of CNS metastases, active autoimmune orinfectious diseases, pregnancy and lactation, as well as significantpsychiatric abnormality. Peptide pulsed dendritic cells were generatedas previously described (82). Briefly, PBMCs from leukapheresis wereisolated on LYMPHOPREP® (density gradient medium) (Nycomed Pharma),frozen in aliquots and stored in liquid nitrogen. One week prior tovaccination, PBMCs were thawed, washed and cultured in medium containinggentamycin, glutamine and heat inactivated autologous plasma. On day 1and 5, IL-4 and GM-CSF were added. To differentiate mature DCs, TNF-γand prostaglandin E2 were added on day 6. On day 7, cells displayingphenotypical and morphological characteristics of mature DCs, i.e. aveiled appearance and =75% CD83 expression, were pulsed with a modifiedsurvivin-derived HLA-A2 restricted survivin₉₆₋₁₀₄ epitope, LMLGEFLKL(SEQ ID NO 10)(Clinalfa, Switzerland) 14. Cells were only used forvaccination if microbial tests of samples taken from cultures on days 1and 5 proved to be sterile.

Patients were vaccinated at 7-day intervals for the first twovaccinations followed by 28-day intervals for further vaccinations. Atotal of 10-20×10⁶ mature, survivin₉₆₋₁₀₄ pulsed DCs were resuspended inPBS, containing 1% human serum albumin, and injected intradermally inaliquots of 1.5×10⁶ DCs per injection site in the ventromedial regionsof the thighs close to the regional lymph nodes. Limbs where draininglymph nodes had been removed and/or irradiated were excluded.Leukapheresis was repeated after 5 vaccinations in absence of severedeterioration of patient's state of health or occurrence of CNSmetastases.

Measurement of Clinical and Immunological Responses

CT scans were performed prior to vaccination and every three monthsthereafter or in case of severe clinical signs of disease progression.Immunological responses were monitored by the ELISPOT assay, using PBMCsobtained every three months, to detect survivin₉₆₋₁₀₄ specific IFN-γrelease. To extend the sensitivity of the ELISPOT assay, PBMCs werestimulated once in vitro at a concentration of 1×10⁶ cells per ml in24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker,Walkersville, Md.), supplemented with 5% heat-inactivated human serumand 2 mM of L-glutamine in the presence of 10 μM of peptide. Two dayslater, 40 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen,Germany) were added. After 10 days the cells were tested for reactivity.To this end, nitrocellulose bottomed 96-well plates (MULTISCREEN® MAIPN45 (96-well assay plate), Millipore, Glostrup, Denmark) were coatedwith an anti-IFN-7 antibody (1-D1K, Mabtech, Sweden). Lymphocytes wereadded at 10⁴-10⁵ cells in 200 μl X-VIVO® medium (chemically defined,serum-free media) per well together with 10⁴ T2-cells and the relevantpeptides at a final concentration of 2 μM. After an overnight incubationat 37° C. and two washes, the biotinylated detection antibody(7-B6-1-Biotin, Mabtech, Sweden) was added; its specific binding wasvisualised using alkaline phosphatase-avidin together with therespective substrate (GibcoBRL). The reaction was terminated upon theappearance of dark purple spots, which were quantitated using the ALPHAIMAGER® System (imaging software) (Alpha Innotech, San Leandro, Calif.,USA).

Survivin96-104/HLA-A*0201 reactive CD8+ T lymphocytes were also trackedin situ both at the vaccination sites as well as in visceral, softtissue, or cutaneous metastases by means of multimericsurvivin96-104/HLA-A*0201 complexes. Vaccination sites were excised 24 hafter intradermal injection in all patients, whereas metastatic lesionswere only removed in selected patients, if easily accessible (patientsKN and GB), or removed during a curative intent (patient WW). Thestaining procedure for multimeric peptide/MHC complexes has beendescribed recently (68). The multimeric survivin96-104/HLA-A*0201complexes were generated by introduction of a recognition site forenzymatic biotinylation at the 5′ end of the extracellular domains ofHLA-A*0201 (residues 1-275). The recombinant protein was purified bysize-exclusion (SEPHADEX G 25® (gel filtration), Pharmacia, Erlangen,Germany) and ion exchange (MONO-Q®, Pharmacia) chromatography and foldedin vitro by dilution in presence of the respective peptides andβ2-microglobulin. After gel filtration on a Sephadex G25® (gelfiltration) column, the protein was multimerized with streptavidin-FITCconjugated to dextran molecules (kindly provided by L. Winther, DAKO,Copenhagen, Denmark) to generate multivalent HLA-dextran complexes.Cryopreserved sections of the respective samples were dried over-nightand subsequently fixed in cold acetone for 5 min. All incubation stepswere performed in the dark at room temperature as follows: (i) 45 min ofan anti-CD8 antibody (1:100, clone HIT8a, Pharmingen, San Diego,Calif.), (ii) Cy3-conjugated goat antimouse (1:500 diluted; code115-165-100, Dianova, Hamburg, Germany) for 45 min and finally (iii) themultimers for 75 min. Between each step the slides were washed twice for10 min in PBS/BSA 0.1%. Finally, slides were mounted in VECTASHIELD®(antifade mounting medium) and observed under a Leica ConfocalMicroscope (TCS 4D, Leica, Mannheim, Germany).

Results

Patient Characteristics, Toxicity and Clinical Course

Five far-advanced stage IV melanoma patients were enrolled, twosuffering from uveal melanoma, one from soft tissue melanoma and theremaining two from cutaneous melanoma. Due to the manifestation ofsymptomatic brain metastases, one patient was taken off therapy afteronly two vaccinations. The other four patients received up to 15vaccinations. One patient died from cardiac arrest in tumor free statusafter surgical resection of remaining metastases. Another patient wastaken off therapy after 10 vaccinations because of appearance ofvisceral metastases (RW). One patient remained on study after 15vaccinations. Detailed patient characteristics, previous therapy, numberof vaccinations and survival status are summarized in table 5.

No major toxicities occurred. Thus, hemoglobin, leucocytes andthrombocytes, as well as lactate dehydrogenase, creatinine andcholinesterase were not influenced by the vaccination therapy (FIG. 16).No signs of systemic or local toxicity were observed at the injectionsites. Furthermore, there was no detection of impaired wound healing,hemorrhagic disorders, cardiac dysfunction, vasculitis or inflammatorybowel disease. In one patient (WW), pre-existing liver metastases couldbe stabilized under vaccination therapy, but a new adrenal metastasisstill occurred. Unfortunately, this patient died due to cardiac arrest,even though tumor-free after curative surgery. A brain metastasis wasdetected in patient PB only 4 weeks after initiation of vaccination.Therefore, this patient had to be excluded from further vaccinationsafter only two DC injections. The other three patients demonstrated slowprogression of metastatic disease without substantial impairment intheir general state of health. Remarkably, for patient KN, an overallsurvival of 13 months (from vaccination start to death) could beachieved despite a heavy metastatic load and fast disease progression atthe start of vaccination. Patient GB remained on protocol 14 monthsafter initiation of vaccination with survivin-peptide pulsed DCs. Itshould be noted, however, that both patients (RW and GB) receivedadditional localized treatment for tumor control, either radiation ofsubcutaneous tumors (RW) or focal chemotherapy (GB).

Survivin-Specific CD8+ T Cell Responses

To monitor the kinetics of cytotoxic T cell responses, PBMCs obtainedprior to and three months after vaccination were tested for reactivityto the modified survivin₉₆₋₁₀₄ epitope by ELISPOT for IFN-γ. Beforeanalysis, PBMCs were stimulated once in vitro to extend the sensitivityof this assay. In all four patients tested, an induction ofsurvivin₉₆₋₁₀₄ reactive T cells was evident (FIG. 17). Analysis forreactivity to other HLA-A*0201 restricted survivin peptides, i.e. thenon-modified survivin₉₆₋₁₀₄ and the adjacent Sur9 epitope, demonstrateda T cell response against these peptides in two of the patients (KN andRW)(data not shown).

The prognostic and clinical value of measurements of tumor-specificT-cell responses in peripheral blood has been questioned repeatedly;thus, we also tested for the presence of survivin₉₆₋₁₀₄/HLA-A*0201reactive CD8+ T lymphocytes among tumor infiltrating lymphocytes in situby peptide/MHC multimer staining. To validate the method, we firstanalyzed tissue samples from delayed type hypersensitivity reactionsoccurring at the vaccination site within 24 hrs. This analysis confirmedearlier observations that intradermal injections of peptide-pulsed DCinduce a strong peptide-specific inflammatory T-cell infiltrate.Subsequently, the peptide/MHC multimer staining procedure was applied onsoft tissue and visceral metastases, which revealed the presence ofsurvivin₉₆₋₁₀₄/HLA-A*0201 reactive cells among the CD8+ infiltrate. Thisobservation suggests that the vaccination does not only induce T cellwith the desired specificity, but also endows them with the necessaryhoming capacity.

TABLE 5 Summary of vaccination trials: patient characteristics Time fromSurvival Patient Age/ primary tumor Measurable No. of after first ID Sexto stage IV Previous therapy disease Clinical outcome vaccinationsvaccination* GB 40/female 4 years, 8 LITT, fotemusine/IL- liver PD (slowgrowth of 15 +14 months months 2IFNα, pre-existing and newtreosulfan/gemcitabine hepatic lesions, new pancreas and pleuralmetastases) KN 53/male 11 years IL-2IFNα/histamine, liver, kidney, PD(slow growth of 13 13 months fotemustine, soft tissue, bone pre-existinglesions, treosulfan/gemcitabine new lymph node, pleural and mediastinallesions) WW 73/male 14 months surgery, DC- liver PD (stable hepatic, 1212 months vaccination, but new adrenal due post- decarbazine metastasis)surgical stroke RW 72/male 16 years surgery, radiotherapy, soft tissuePD (growth of pre- 12 +12 months adriblastin/ifosfamid, existing and newsoft ixoten, decarbazine, tissue metastases; TNF/melphalan detection ofheart, lung and muscle metastases after 12 vaccinations) PB 52/male 2years, 3 radiotherapy lung, kidney PD (new skin and 2 4 months monthsbrain metastases)

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The invention claimed is:
 1. An immunogenic composition consisting ofthe peptide of the amino acid sequence of SEQ ID NO: 18 and an adjuvant.2. An immunogenic composition consisting of the peptide of the aminoacid sequence of SEQ ID NO: 18 coupled to an immunogenic carrier, and anadjuvant.
 3. An immunogenic composition consisting of the peptide of theamino acid sequence of SEQ ID NO: 18, an adjuvant, and at least one of apharmaceutical carrier, diluent, preservative, buffer component, or acombination thereof.